Oncostatin m (osm) antagonists for preventing cancer metastasis and il-6 related disorders

ABSTRACT

A method of treating cancer or metastasis is provided involving administering at least one oncostatin M (OSM) antagonist to a subject, wherein the subject has been diagnosed with cancer. Administration of an OSM antagonist such as a small molecule pharmaceutical is provided as well as an anti-OSM antibody, an anti-OSM aptamer, and an 
     OSM mRNA antagonist. The OSM antagonists were found to inhibit or prevent tumor cell detachment, proliferation and metastasis in several cancer types.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. Ser. No. 14/478,175, filedSep. 5, 2014, which claims priority under 35 U.S.C. §119 to provisionalapplications U.S. Ser. No. 61/874,044 and U.S. Ser. No. 61/874,181, bothfiled Sep. 5, 2013, herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure generally relates to treatment of cancer, and,more specifically, to the inhibition of signaling molecules implicatedin the invasion and metastasis of cancer cells.

BACKGROUND OF THE INVENTION

Metastasis or metastatic disease is the spread of a disease from oneorgan or part to another non-adjacent organ or part. Metastatic diseaseis primarily but not uniquely associated with malignant tumor cells andinfections relating to cancer. (Klein, 2008, Science 321(5897):1785-88;Chiang & Massague, 2008, New Engl. J. Med. 359(26):2814-23).

Cancer occurs after a single cell in a tissue is genetically damaged inways that result in the formation of a putative cancer stem cellpossessing a malignant phenotype. These cancer stem cells are able toundergo uncontrolled abnormal mitosis, which serves to increase thetotal number of cancer cells at that location. When the area of cancercells at the originating site become clinically detectable, it is calledprimary tumor. Some cancer cells also acquire the ability to penetrateand infiltrate surrounding normal tissues in the local area, forming anew tumor. The newly formed tumor in the adjacent site within the tissueis called a local metastasis.

Some cancer cells acquire the ability to penetrate the walls oflymphatic and/or blood vessels, after which they are able to circulatethrough the bloodstream (circulating tumor cells) to other sites andtissues in the body. This process is known (respectively) as lymphaticor hematogenous spread. After the tumor cells come to rest at anothersite, they re-penetrate through the vessel or walls (extravasion),continue to multiply, and eventually another clinically detectable tumoris formed. This new tumor is known as a metastatic (or secondary) tumor.Metastasis is one of the hallmarks of malignancy. Most tumors and otherneoplasms can metastasize, although in varying degrees (e.g. basal cellcarcinoma rarely metastasizes) (Kumar et al., 2005, “Robbins and CotranPathologic Basis of Disease”, 7th ed., Philadelphia: Elsevier Saunders).

Metastatic tumors are very common in the late stages of cancer. The mostcommon places for the metastases to occur are the lungs, liver, brain,and the bones. There is also a propensity for a tumor to seed inparticular organs. For example, prostate cancer usually metastasizes tothe bones. In a similar manner, colon cancer has a tendency tometastasize to the liver. Stomach cancer often metastasizes to theovaries in women. Breast tumor cells often metastasize to bone tissue.Studies have suggested that these tissue-selective metastasis processesare due to specific anatomic and mechanical routes.

Oncostatin M (OSM) is a 28 kDa multifunctional member of the IL-6 familyof cytokines secreted by monocytes, macrophages, neutrophils andactivated T-lymphocytes (Tanaka & Miyajima, Rev Physiol BiochemPharmacol 149: 39-53, 2003). Proteolytic cleavage near thecarboxy-terminus of the secreted OSM yields the fully active form ofOSM, 209 amino acids length having two N-linked glycosylation sites. OSMbelongs to the IL-6 family of cytokines that includes (IL-6, IL-11,leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neutotrophicfactor (CNTF) and cardiotrophin-like cytokine

(CLC)) which share a common receptor subunit, gp130 protein. In humans,OSM signals through receptor heterodimers consisting of gp130 and theLIFRα subunit or gp130 and the OSMRβ subunit. In contrast to the othercytokines of the IL-6 family, OSM binds gp130 directly and in theabsence of any additional membrane-bound co-receptor (Gearing et al.,Science 255: 1434-1437, 1992). Following OSM binding to gp130, OSMRβ orLIFRα are recruited to form a high-affinity signaling complex (Mosley etal., J Biol. Chem. 271: 32635-32643, 1996). Activation of eitherreceptor results in signaling via the JAK/STAT pathway (Auguste et al.,J Biol. Chem. 272: 15760-15764, 1997).

OSM is produced primarily by cells of immune system origin and, has beena target for diseases associated with autoimmune disorders.

It is an object of the present in invention to provide novel OSMantagonist pharmaceutical compositions and their use in preventingcancer metastasis.

SUMMARY OF THE INVENTION

To achieve these and other advantages, and in accordance with thepurposes of the present invention, as embodied and broadly describedherein, the present invention relates to a method of reducing tumor celldetachment, proliferation and/or metastasis involving administering atleast one OSM antagonist or OSM receptor antagonist to a subject. In apreferred embodiment the tumor cells are those associated with prostateor breast cancer.

Any number of OSM or OSM receptor antagonists may be employed, alone, orin combination with other therapies for treating or reducing metastasisand tumor cell migration. OSM antagonists may include small molecules,antibodies, antibody-derived reagents, or chimeric molecules. Includedin the definition of antagonist is a structural or functional mimetic ofany such molecule described above. Also contemplated are nucleic acidmolecules such as DNA or RNA aptamers.

The antagonist may function by blocking OSM from interaction with theOSM receptor gp130, or the other OSM receptors, OSMrβ chain or LIFr, orby blocking formation of heterodimers of these proteins, and as suchprevent OSM binding and signaling thereby reducing synthesis ofcytokines and/or matrix metalloproteinases (MMPs). The antagonistaccording to the invention may therefore be a ligand for either OSM orone or more of the OSM receptors (gp130, OSMrβ or LIFr) or an agentcapable of interfering with these interactions in a manner which affectsOSM biological activity. Hereinafter reference to an antagonist to OSMcan be taken to mean either an antagonist to OSM itself or to one of itsreceptors.

Administration of at least one OSM or OSM receptor antagonist, alone, orin combination with one or more therapies can be administered to apatient in need thereof to reduce tumor cell detachment, proliferationand/or metastasis.

One embodiment provides a pharmaceutical composition including an OSMantagonist in an amount effective to inhibit or reduce tumor celldetachment, proliferation and/or metastasis.

Another embodiment provides methods for inhibiting tumor celldetachment, proliferation and/or metastasis by administering an OSMantagonist. Preferably, the OSM antagonist specifically binds to OSM orthe OSM receptor and inhibits or reduces OSM biological activity.

Another embodiment provides methods for identifying inhibitors of OSM.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are included to illustrate certain aspects of thepresent disclosure, and should not be viewed as exclusive embodiments.The subject matter disclosed is capable of considerable modifications,alterations, combinations, and equivalents in form and function, as willoccur to those skilled in the art and having the benefit of thisdisclosure.

FIG. 1A. OSM is present at high levels in tissues with ductal carcinomain situ and invasive ductal carcinoma, as compared to adjacent normalbreast and metastatic tissues by immunohistochemistry. Human breasttissue (adjacent normal, ductal carcinoma in situ (DCIS), and invasiveductal carcinoma (IDC)) were stained with 1:400 dilution of hOSMantibody. Secondary antibody (rabbit anti-goat) alone served as anegative control. OSM is visualized by red-brown staining. Images weretaken using 100× and 400× magnification with a light microscope. FIG. 1BOSM is highly expressed in the primary mammary tumor, and recruitedcells of the lung microenvironment. Histology using H&E confirms thepresence of a large, primary mammary tumor (T) 32 days after tumor cellinjection into the 4^(th) mammary fat pad of female Balb/c mice and highOSM expression in the tumor as well as background expression in thenormal breast connective tissue (CT). OSM expression is shown to behighest in the invasive edge of the tumor (T) closest to the normalbreast connective tissue (CT). Control slides with no primary OSMantibody show low background staining.

FIGS. 2A-2B. The number of metastases was reduced by more than 50% inmice injected with 4T1.2-shOSM2 cells compared to control 4T1.2-LacZcontrol cells at both mid and late stages of metastasis. FIG. 2B showshistology slides.

FIGS. 3A-3C. Reduced OSM expression results in fewer spontaneous lungmetastases and lower total volume of lung metastases by MRI. FIG. 3A,spontaneous lung metastases are more frequently detected by MRIbeginning at day 20-21 until the endpoint of the experiment 29-30 ofmice orthotopically injected with parental 4T1.2 or control 4T1.2-LacZcells as compared to 4T1.2-shOSM2 cells. FIG. 3B, quantification of thetotal number and FIG. 3C, volume of lung metastases shows significantlyhigher lung metastases in the 4T1.2 or 4T1.2-LacZ injected mice ascompared to the 4T1.2-shOSM2 injected mice. (4T1.2, n=6; 4T1.2-LacZ,n=7; 4T1.2-shOSM2, n=7). Data expressed as mean ±SEM, *P<0.05, **P<0.01,t-test.

FIGS. 4A-4C. Reduced OSM expression by 4T1.2 tumors increases survival.FIG. 4A, the timeline shows orthotopic mammary tumor cell injection atday 0, resection at day 14, and final day of sacrifice per group.Kaplan-Meier analysis of survival following tumor resection shows asignificant increase in survival in mice injected with 4T1.2-shOSM1cells and 4T1.2-shOSM2 cells as compared to control 4T1.2-LacZ cells.FIG. 4B, the timeline shows intracardiac mammary tumor cells injectionat day 0 and final day of sacrifice. Kaplan-Meier analysis of survivalshows no difference in survival in mice injected with control 4T1.2-LacZand 4T1.2-shOSM2 cells and FIG. 4C, also no difference in the amount oflung metastases quantified by qPCR. (4T1.2-LacZ, n=8-9; 4T1.2-shOSM1,n=7; 4T1.2-shOSM2, n=9-12) Data expressed as mean±SEM. *P<0.05,***P<0.001, Log-rank test.

FIGS. 5A and 5B show plots of decreased detached 4T1.2 mammary tumorcells and 4T1.2 cell migration with OSM treatment (25 ng/ml).

FIGS. 6A-6F show human MDA-MB-231 D3H2LN cells in vivo. FIG. 6A. Thetimeline shows orthotopic mammary tumor cell injection at day 0,peri-tumoral OSM or PBS injections beginning 3× per week at day 13, andfinal day of sacrifice of both groups at day 61. FIGS. 6B. MDA-MB-231D3H2LN luc2 cells were injected into the fourth mammary fat pad offemale nude mice and tumor growth was measured using calipers. Averagetumor volume (mm³) did not differ between the peri-tumorally injectedOSM and PBS control groups. FIG. 6C and 6D. Representative images of PBSand OSM injected tumor-bearing mice imaged ventrally by BLI. Tumors withhigh BLI produced background on adjacent mice. This background islabeled with *. Ex vivo lungs from mice bearing MDA-MB-231 D3H2LN luc2tumors injected peri-tumorally with PBS or OSM. Note the difference inBLI intensity between the groups. Bioluminescence intensities werequantified ex vivo in the lung. Lungs from mice receiving peri-tumoralOSM injections showed a 200 fold higher BLI intensity as compared to PBSinjections. Data expressed as photon/s (mean±SEM; n=5-6), and expressedgraphically is shown in FIGS. 6E and 6F.

FIG. 7 shows a plot of increased IL-6 expression in the presence of OSM.IL-6 levels were measured by ELISA on conditioned media from various OSMtreated cells. ER negative MDA-MB-231, MDA-MB468, and 4T1.2 cellsdisplayed high levels of induction (4-5 fold), while IL-6 levels did notchange in the ER positive cell lines T47D and MCF7.

FIG. 8 shows a schematic comparing orthotopic and intracardiacinjection. OSM's prometastatic effects occur early in the metastaticcascade. OSM increases detachment, EMT, angiogenesis through VEGF andHIF1a, invasion, proteases, and may subsequently increase intravasationinto the blood vessels. OSM does not have any effect on extravasationand colonization at a secondary site.

FIGS. 9A and 9B show a plot of demonstrating how OSM promotes metastasesto the lung and other organs in a 4T1.2 orthotopic mouse model.

FIG. 10 shows a schematic of the mechanism by which OSM is presumed topromote the metastasis and progression of prostate tumors.

FIGS. 11A and 11B show proliferation for Du145 and PC3 prostate cancercell lines. FIG. 11A. The prostate cancer cell line Du-145 showsincreased proliferation after 5 days of treatment with OSM (17.5 ng/ml)compared to untreated cells. FIG. 11B. The prostate cancer cell linePC-3 did not show increased proliferation in response to OSM treatment(17.5 ng/ml).

FIGS. 12A and 12B show detachment Changes for the DU-145 and PC-3Prostate Cancer Cell Lines. FIG. 12A. The prostate cancer cell lineDu-145 shows increased detachment after 5 days of treatment with OSM(17.5 ng/ml) compared to untreated cells. FIG. 12B. The prostate cancercell line PC-3 did not show increased detachment in response to OSMtreatment (17.5 ng/ml).

FIG. 13. Morphology changes for DU-145 prostate cancer cell line. After5 days of OSM treatment (17 .5 ng/ml), DU-145 cells exhibit amesenchymal orphology characteristic of having undergone EMT. Untreatedprostate cancer cells exhibit a more epithelial morphology and form moretightly packed colonies.

FIG. 14. Invasive Potential for the DU-145 and PC-3 Prostate Cancer CellLines. Both the DU-145 and PC-3 cell lines show an increased invasionpotential as demonstrated by Matrigel assays after 24 hours of OSMtreatment (17.5 ng/ml).

FIGS. 15A and 15B. OSM-SMI-8 decreases OSM-induced detachment in humanprostate cancer cells. Detachment for FIG. 15A) Du145 and 15B) PC3+STAT3human prostate cancer cells. 10,000 cells were plated in a 24-well platein 1 mL complete media that was pre-incubated with OSM (5 ng/mL) andinhibitors (5 μM) for 1 hour. After 5 days, the detached cells werestained with Trypan Blue and counted using a hemocytometer. OSM-SMI-1does not decrease detachment, while OSM-SMI-8, -10, and -11 decreaseOSM-induced detachment.

FIGS. 16A-16D. OS-induced pSTAT3 is blocked by OSM-SMI-8. FIG. 16A,human breast cancer cells treated with OSM (25 ng/ml) induce pSTAT3expression as measured by ELISA. Neutralizing antibodies to OSM or gp130attenuate pSTAT3 levels. This method can be used for in vitro screeningof lead compounds that inhibit OSM signaling. (mean±SEM; n=3 ; p<0.001between +OSM versus either anti-OSM or anti-gp130, unpaired t-test) FIG.16B, T47D human breast cancer cells, FIG. 16C, MDA-MB-231 human breastcancer cells, and FIG. 16D, Du145 human prostate cancer cells werepre-treated with OSM-SMI-1 to -16 (Table 1; 5 μM) for 2 hours and thentreated with OSM (5 ng/ml) for 30 minutes. Cell lysates were collectedand pSTAT3 levels were measure by ELISA. OSM-SMI-8 inhibition of pSTAT3suggests decreased OSM signaling.

FIGS. 17A and 17B show OSM-SMI-8 inhibits OSM signaling in humanMDA-MB-231 cells. FIG. 17A, an IC₅₀ concentration of 531 μM wasdetermined for MDA-MB-231 cells blocked with OSM-SMI-8 (and then treatedwith OSM (5 ng/ml) for 30 minutes. FIG. 17B, three OSM-SMIs wereassessed in two independent sets of MDA-MB-231 cell lysates by Westernblot analysis for suppression of downstream pSTAT3, pJNK, pERK, and pAKTsignaling. STAT3 and actin protein levels were used as internal loadingcontrols.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure generally relates to treatment of cancer, and,more specifically, to the inhibition of signaling molecules implicatedin the invasion and metastasis of cancer.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the present specification and associated claims areto be understood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification are approximations that mayvary depending upon the desired properties sought to be obtained by thepresent embodiments. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaim, each numerical parameter should at least be construed in light ofthe number of reported significant digits and by applying ordinaryrounding techniques.

One or more illustrative embodiments incorporating the featuresdisclosed herein are presented below. Not all features of a physicalimplementation are necessarily described or shown in this applicationfor the sake of clarity. It is to be understood that in the developmentof a physical embodiment incorporating the embodiments of the presentdisclosure, numerous implementation-specific decisions can be made toachieve the developer's goals, such as compliance with system-related,business-related, government-related and other constraints, which varyby implementation and from time to time. While a developer's effortsmight be time-consuming, such efforts would be, nevertheless, a routineundertaking for those of ordinary skill the art and having benefit ofthis disclosure.

While compositions and methods are described herein in terms of“comprising” various components or steps, the compositions and methodscan also “consist essentially of” or “consist of” the various componentsand steps.

As used herein, the term “prevent” or “prevention” means no disorder ordisease development if none had occurred, or no further disorder ordisease development if there had already been development of thedisorder or disease. Also considered is the ability of one to preventsome or all of the symptoms associated with the disorder or disease.Disease and disorder are used interchangeably herein.

The terms “inhibit” and “antagonize”, as used herein, mean to reduce amolecule, a reaction, an interaction, a gene, an mRNA, and/or aprotein's expression, stability, function or activity by a measurableamount or to prevent entirely. Inhibitors are compounds that, e.g., bindto, partially or totally block stimulation, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate a protein, a gene,and an mRNA stability, expression, function and activity, e.g.,antagonists.

As used herein, the terms “effective amount” or “therapeuticallyeffective amount” or “pharmaceutically effective amount” of a compoundare used interchangeably to refer to the amount of the compound which issufficient to provide a beneficial effect to the subject to which thecompound is administered. The term to “treat,” as used herein, meansreducing the frequency with which symptoms are experienced by a patientor subject or administering an agent or compound to reduce the severitywith which symptoms are experienced. An appropriate therapeutic amountin any individual case may be determined by one of ordinary skill in theart using routine experimentation. By the term “specifically bind” or“specifically binds,” as used herein, is meant that a first molecule(e.g., an antibody) preferentially binds to a second molecule (e.g., aparticular antigenic epitope), but does not necessarily bind only tothat second molecule.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the term “pharmaceutical composition” refers to amixture of at least one compound useful within the invention with otherchemical components, such as carriers, stabilizers, diluents, dispersingagents, suspending agents, thickening agents, and/or excipients. Thepharmaceutical composition facilitates administration of the compound toan organism. Multiple techniques of administering a compound exist inthe art including, but not limited to: intravenous, oral, aerosol,parenteral, ophthalmic, pulmonary and topical administration.

The language “pharmaceutically acceptable carrier” includes apharmaceutically acceptable salt, pharmaceutically acceptable material,composition or carrier, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting a compound(s) of the present invention within or to thesubject such that it may perform its intended function. Typically, suchcompounds are carried or transported from one organ, or portion of thebody, to another organ, or portion of the body. Each salt or carriermust be “acceptable” in the sense of being compatible with the otheringredients of the formulation, and not injurious to the subject. Someexamples of materials that may serve as pharmaceutically acceptablecarriers include: sugars, such as lactose, glucose and sucrose;starches, such as corn starch and potato starch; cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients,such as cocoa butter and suppository waxes; oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; glycols, such as propylene glycol; polyols, such asglycerin, sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; diluent; granulating agent; lubricant; binder;disintegrating agent; wetting agent; emulsifier; coloring agent; releaseagent; coating agent; sweetening agent; flavoring agent; perfumingagent; preservative; antioxidant; plasticizer; gelling agent; thickener;hardener; setting agent; suspending agent; surfactant; humectant;carrier; stabilizer; and other non-toxic compatible substances employedin pharmaceutical formulations, or any combination thereof. As usedherein, “pharmaceutically acceptable carrier” also includes any and allcoatings, antibacterial and antifungal agents, and absorption delayingagents, and the like that are compatible with the activity of thecompound, and are physiologically acceptable to the subject.Supplementary active compounds may also be incorporated into thecompositions.

As used herein, the term “antagonist” generally refers to the propertyof a molecule, compound or other agent to, for example, interfere withthe binding of one molecule with another molecule or the stimulation ofone cell by another cell either through steric hindrance, conformationalalterations or other biochemical mechanisms. In one regard, the termantagonist relates to the property of an agent to prevent the binding ofa receptor to its ligand, e.g., the binding of OSM with gp 130 or otherOSM receptors, thereby inhibiting the signal transduction pathwaytriggered by OSM. The term antagonist is not limited by any specificaction mechanism, but, rather, refers generally to the functionalproperty presently defined. Antagonists of the present inventioninclude, but are not limited to: small molecules and chemical compoundsthat bind to OSM or one of its receptors as well as OSM antibodies andfragments, muteins, and modifications thereof, peptides, and nucleicacid molecules such as antisense or RNAi compounds that inhibitexpression of OSM.

As used herein, the term “heavy chain antibody” or “heavy chainantibodies” comprises immunoglobulin molecules derived from camelidspecies, either by immunization with an antigen and subsequent isolationof sera, or by the cloning and expression of nucleic acid sequencesencoding such antibodies. The term “heavy chain antibody” or “heavychain antibodies” further encompasses immunoglobulin molecules isolatedfrom an animal with heavy chain disease, or prepared by the cloning andexpression of V_(H) (variable heavy chain immunoglobulin) genes from ananimal.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

By the term “applicator,” as the term is used herein, is meant anydevice including, but not limited to, a hypodermic syringe, a pipette,and the like, for administering the compounds and compositions of theinvention.

As used herein, “aptamer” refers to a small molecule that can bindspecifically to another molecule. Aptamers are typically eitherpolynucleotide- or peptide-based molecules. A polynucleotidal aptamer isa DNA or RNA molecule, usually comprising several strands of nucleicacids, that adopt highly specific three-dimensional conformationdesigned to have appropriate binding affinities and specificitiestowards specific target molecules, such as peptides, proteins, drugs,vitamins, among other organic and inorganic molecules. Suchpolynucleotidal aptamers can be selected from a vast population ofrandom sequences through the use of systematic evolution of ligands byexponential enrichment. A peptide aptamer is typically a loop of about10 to about 20 amino acids attached to a protein scaffold that bind tospecific ligands. Peptide aptamers may be identified and isolated fromcombinatorial libraries, using methods such as the yeast two-hybridsystem.

“Naturally-occurring” as applied to an object refers to the fact thatthe object can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man is a naturally-occurring sequence.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “epitope” as used herein is defined as a small chemicalmolecule on an antigen that can elicit an immune response, inducing Band/or T cell responses. An antigen can have one or more epitopes. Mostantigens have many epitopes; i.e., they are multivalent. In general, anepitope is roughly five amino acids and/or sugars in size. One skilledin the art understands that generally the overall three-dimensionalstructure, rather than the specific linear sequence of the molecule, isthe main criterion of antigenic specificity and therefore distinguishesone epitope from another.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds.Synthetic polypeptides can be synthesized, for example, using anautomated polypeptide synthesizer.

The term “protein” typically refers to large polypeptides. The term“peptide” typically refers to short polypeptides. Conventional notationis used herein to portray polypeptide sequences: the left-hand end of apolypeptide sequence is the amino-terminus; the right-hand end of apolypeptide sequence is the carboxyl-terminus.

As used herein, a “peptidomimetic” is a compound containing non-peptidicstructural elements that is capable of mimicking the biological actionof a parent peptide. A peptidomimetic may or may not comprise peptidebonds.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the compositionand/or compound of the invention in a kit. The instructional material ofthe kit may, for example, be affixed to a container that contains thecompound and/or composition of the invention or be shipped together witha container which contains the compound and/or composition.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the recipient uses theinstructional material and the compound cooperatively. Delivery of theinstructional material may be, for example, by physical delivery of thepublication or other medium of expression communicating the usefulnessof the kit, or may alternatively be achieved by electronic transmission,for example by means of a computer, such as by electronic mail, ordownload from a website.

Throughout this disclosure, various aspects of the invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

I). OSM Antagonists

The antagonist may function by blocking OSM from interaction with theOSM receptor gp130, or the other OSM receptors, OSMrβ chain or LIFr, orby blocking formation of heterodimers of these proteins, and as suchprevent OSM binding and signaling thereby reducing synthesis ofcytokines and/or MMPs. The antagonist according to the invention maytherefore be a ligand for either OSM or one or more of the OSM receptors(gp130, OSMrβ or LIFr) or an agent capable of interfering with theseinteractions in a manner which affects OSM biological activity.Hereinafter reference to an antagonist to OSM can be taken to meaneither an antagonist to OSM itself or to one of its receptors.

Nucleic acid and amino acid sequences are known and generally availableto those of skill in the art at places such as Gen bank, Swiss Prot, andEMBL. They are also disclosed herein as SEQ ID NOS:1 and 2. Further,amino acid residues which are important for OSM's interaction with gp130have been identified. From the published amino acid sequence of OSM(Malik et al., 1989, Mol. Cell. Biol., 9(7), 2847-53, DNA sequence entryM27288 in EMBL database, protein sequence entry P13725 in Swissprot)these are G120, Q16 and Q20; N123 and N124 may also play a part. Thefirst 25 residues are a signal peptide, and the mature protein begins atthe sequence AAIGS. The sequence is numbered from the first amino acidof the mature protein as shown ion SEQ ID NO: 1.

The invention therefore further provides an antagonist or agent capableof interacting with one or more of these specific residues and/or thebinding sites they help to define on OSM to alter OSM biologicalactivity.

Potential antagonists of OSM include small organic molecules, ions whichinteract specifically with OSM for example a substrate possibly anatural substrate, a cell membrane component, a receptor or a naturalligand, a fragment thereof or a peptide or other proteinaceous molecule,particularly preferred is a non-signaling mutant form of OSM which willblock binding of OSM to the OSM receptor, but also modified OSMmolecules. Such antagonists may be in the form of DNA encoding theprotein or peptide and may be delivered for in vivo expression of saidantagonist. Antagonists may be vaccines comprising such protein orpeptide molecules or DNA, designed to produce an antagonistic effecttowards OSM via induction of antibody responses in vivo targeted towardsnative OSM. Such antagonists may also include antibodies,antibody-derived reagents or chimeric molecules. Included in thedefinition of antagonist is a structural or functional mimetic of anysuch molecule described above. Also contemplated are nucleic acidmolecules such as DNA or RNA aptamers.

OSM antagonists of the present invention include, where applicable,functional equivalents. For example, molecules may differ in length,structure, components, etc., but may still retain one or more of thedefined functions. Preferred OSM antagonist small molecules may bemodified to include side groups, or other chemical additions which donot affect the antagonist activity.

Further, functional equivalents of the antibodies, antibody fragments orpeptides of the present invention may include mimetic compounds, i.e.,constructs designed to mimic the proper configuration and/or orientationfor antigen binding.

OSM antagonists may optionally be modified by addition of side groups,etc., e.g., by amino terminal acylation, carboxy terminal amidation orby coupling of additional groups to amino acid side chains. Antagonistsmay also comprise one or more conservative amino acid substitutions. By“conservative amino acid substitutions” is meant those changes in aminoacid sequence that preserve the general charge,hydrophobicity/hydrophilicity and/or steric bulk of the amino acidsubstituted. For example, substitutions between the following groups areconservative: Gly/Ala, Val/Ile/Leu, Asp/Glu, Lys/Arg, Asn/Gln,Ser/Cys/Thr, and Phe/Trp/Tyr. Such modifications will not substantiallydiminish the efficacy of the OSM antagonists and may impart such desiredproperties as, for example, increased in vivo half-life or decreasedtoxicity.

The invention is also intended to include polypeptides bearingmodifications other than the insertion, deletion, or substitution ofamino acid residues. By way of example, the modifications may becovalent in nature, and include for example, chemical bonding withpolymers, lipids, other organic, and inorganic moieties. Suchderivatives may be prepared to increase circulating half-life of apolypeptide, or may be designed to improve targeting capacity for thepolypeptide to desired cells, tissues, or organs. Similarly, theinvention further embraces OSM or OSMR polypeptides that have beencovalently modified to include one or more water soluble polymerattachments such as polyethylene glycol, polyoxyethylene glycol, orpolypropylene glycol.

A. OSM Small Molecules

Preferred antagonists include small organic molecules. Such compoundsmay be from any class of compound but will be selected on the basis oftheir ability to affect the biological activity of OSM through one ofthe mechanisms described above and will be physiologically acceptable(non-toxic or demonstrating an acceptable level of toxicity or otherside-effects). One class of compounds which may provide usefulantagonists are ribonucleosides such asN-(1H-pyrazolo[3,4-d]pyrimidin-4-yl) benzamide); Davoll and Kerridge, J.Chem Soc., 2589, 1961). According to the invention, OSM small moleculeantagonists include those listed herein in Table 1 and Table 2. Anystereochemistry depicted in the formula's in Tables 1 and 2 is intendedto be informative only and non-limiting. Any compound with the samebasic chemical formula is intended to be included as are derivatives,isomers, and modifications.

TABLE 1 AutoDock Predicted Binding OSM Free Binding Energy ConstantChemical Class Structure Compound ID (kcal/mol) (uM) bis(benzimidazole)(1)

NCI 61610 −9.33 0.14 spirocyclopropane (2)

CB_CL 181230 −8.56 0.53 spirocyclopropane (3)

CB_CL 81250 −8.39 0.71 spirocyclopropane (4)

CB_CL 111696 −8.54 0.55 quinolone (5)

Florida ZINC 15777366 −8.27 0.87 spirocyclopropane (6)

CB_CL 19531 −8.44 0.65 dihydronaphthalene- 2-one (7)

Florida ZINC 15770835 −7.47 3.35 benzochromene (8)

Florida ZINC 32603276 −7.53 3.02 naphthyridine-2-one (9)

CB_CL 12813 −7.30 4.46 phenanthridine-6- one (10)

NCI 127133 −7.12 6.04

TABLE 2 AutoDock Predicted Binding Binding Compound Compound Free EnergyConstant Number ID (kcal/mol) (uM) OSM-SMI-1  NSC21357

−6.88 9.11 OSM-SMI-2  NSC81514

−6.04 27.52 OSM-SMI-3  NSC105360

−8.45 0.64 OSM-SMI-4  NSC112821

−5.81 55.05 OSM-SMI-5  NSC348965

−7.93 1.53 OSM-SMI-6  NSC382916

−8.99 0.26 OSM-SMI-7  NSC636120

−8.26 0.88 OSM-SMI-8  NSC642624

−7.56 2.87 OSM-SMI-9  NSC645072

−7.03 7.05 OSM-SMI-10 NSC647257

−9.86 0.06 OSM-SMI-11 NSC648596

−5.87 50.04 OSM-SMI-12 NSC127133

−7.12 6.04 OSM-SMI-13 NSC61610

−9.33 0.14 OSM-SMI-14 CB_CL111696

−8.54 0.55 OSM-SMI-15 CB_CL19531

−8.44 0.65 OSMI-SMI-16 CB_CL81250

−8.39 0.71

B. OSM Antibodies

The term “antibody” is used in the broadest-sense and includes fullyassembled antibodies, monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), antibodyfragments that can bind antigen (e.g., Fab′, F′(ab)2, Fv, single chainantibodies, diabodies), and recombinant peptides comprising the forgoingas long as they exhibit the desired biological activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that are typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. In addition to their specificity, themonoclonal antibodies are advantageous in that they are synthesized bythe homogeneous culture, uncontaminated by other immunoglobulins withdifferent specificities and characteristics.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature,256:495 [1975], or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature, 352:624628[1991] end Marks et al., J. Mol.Biol., 222.1581-597 (1991), for example.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes, IgA, IgD, IgE, IgG and IgM, and several ofthese may be further divided into subclasses or isotypes, e.g. IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains thatcorrespond to the different classes of immunoglobulins are called alpha,delta, epsilon, gamma- and mu respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known. Different isotypes have different effector functions;for example, IgG1 and IgG3 isotypes have ADCC activity.

In certain embodiments, the monoclonal, human, humanized, HumanEngineered™ or variant anti-OSM antibody is an antibody fragment, suchas an RX1, 5H4, MC1, or MC3 antibody fragment. Various techniques havebeen developed for the production of antibody fragments. Traditionally,these fragments were derived via proteolytic digestion of intactantibodies (see, e.g., Morimoto et al., Journal of Biochemical andBiophysical Methods 24:107-117 (1992) and Brennan et al., Science 229:81(1985)). However, these fragments can now be produced directly byrecombinant host cells. Better et al., Science 240: 1041-1043 (1988)disclose secretion of functional antibody fragments from bacteria (see,e.g., Better et al., Skerra et al., Science 240: 1038-1041 (1988)). Forexample, Fab′-SH fragments can be directly recovered from E. coli andchemically coupled to form F(ab′)2 fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). In another embodiment, the F(ab′)2 isformed using the leucine zipper GCN4 to promote assembly of the F(ab′)2molecule. According to another approach, Fv, Fab or F(ab′)2 fragmentscan be isolated directly from recombinant host cell culture. Othertechniques for the production of antibody fragments will be apparent tothe skilled practitioner.

An “isolated” antibody is one that has been identified and separated andfor recovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would interferewith diagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or nonproteinaceous solutes.In preferred embodiments, the antibody will be purified (1) to greaterthan 95% by weight of antibody as determined by the Lowry method, andmost preferably more than 99% by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

For a detailed description of the structure and generation ofantibodies, see Roth, D. B., and Craig, N. L., Cell, 94:411-414 (1998),and U.S. Pat. No. 6,255,458, herein incorporated by reference in itsentirety. Briefly, the process for generating DNA encoding the heavy andlight chain immunoglobulin genes occurs primarily in developing B-cells.Prior to the rearranging and joining of various immunoglobulin genesegments, the V, D, J and constant (C) gene segments are found generallyin relatively close proximity on a single chromosome. DuringB-cell-differentiation, one of each of the appropriate family members ofthe V, D, J (or only V and J in the case of light chain genes) genesegments are recombined to form functionally rearranged heavy and lightimmunoglobulin genes. This gene segment rearrangement process appears tobe sequential. First, heavy chain D-to-J joints are made, followed byheavy chain V-to-DJ joints and light chain V-to-J joints.

As provided herein, the compositions for and methods of treating orpreventing tumor cell detachment, proliferation and/or metastasis mayutilize one or more antibody used singularly or in combination withother therapeutics to achieve the desired effects. Antibodies accordingto the present invention may be isolated from an animal producing theantibody as a result of either direct contact with an environmentalantigen or immunization with the antigen. Alternatively, antibodies maybe produced by recombinant DNA methodology using one of the antibodyexpression systems well known in the art (See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988)).Such antibodies may include recombinant IGs, chimeric fusion-proteinshaving immunoglobulin derived sequences or “Human Engineered” antibodiesthat may all be used for the treatment and prevention of tumor celldetachment, proliferation and/or metastasis according to the presentinvention. In addition to intact, full-length molecules, the term“antibody” also refers to fragments thereof (such as, e.g., scFv, Fv,Fd, Fab, Fab′ and F(ab)′2 fragments) or multimers or aggregates ofintact molecules and/or fragments that bind to OSM (or OSMR). Theseantibody fragments bind antigen and may be derivatized to exhibitstructural features that facilitate clearance and uptake, e.g., byincorporation of galactose residues.

In one embodiment of the present invention, OSM monoclonal antibodiesmay be prepared essentially as described in Halenbeck et al. U.S. Pat.No. 5,491,065 (1997), incorporated herein by reference. Exemplary OSMmonoclonal antibodies include those that bind to an apparentconformational epitope associated with recombinant or native dimeric OSMwith concomitant neutralization of biological activity. These antibodiesare substantially unreactive with biologically inactive forms of OSMincluding monomeric and chemically derivatized dimeric OSM.

In other embodiments of the present invention, Human Engineered anti-OSMmonoclonal antibodies are provided. The phrase “Human Engineeredantibody” refers to an antibody derived from a non-human antibody,typically a mouse monoclonal antibody. Alternatively, a Human Engineeredantibody may be derived from a chimeric antibody that retains orsubstantially retains the antigen binding properties of the parental,non-human, antibody but which exhibits diminished immunogenicity ascompared to the parental antibody when administered to humans. Thephrase “chimeric antibody,” as used herein, refers to an antibodycontaining sequence derived from two different antibodies (see, e.g.,U.S. Pat. No. 4,816,567) which typically originate from differentspecies. Most typically, chimeric antibodies comprise human and murineantibody fragments, generally human constant and mouse variable regions.

The phrase “complementarity determining region” or the term “CDR” refersto amino acid sequences which together define the binding affinity andspecificity of the natural Fv region of a native immunoglobulin bindingsite (See, e.g., Chothia et al., J. Mol. Biol. 196:901 917 (1987); Kabatet al., U.S. Dept. of Health and Human Services NIH Publication No. 913242 (1991)). The phrase “constant region” refers to the portion of theantibody molecule that confers effector functions. In the presentinvention, mouse constant regions are preferably substituted by humanconstant regions. The constant regions of the subject antibodies arederived from human immunoglobulins. The heavy chain constant region canbe selected from any of the five isotypes: alpha, delta, epsilon, gammaor mu.

The antibodies of the present invention are said to be immunospecific orspecifically binding if they bind to antigen with a Ka of greater thanor equal to about 10⁶ M⁻¹ preferably greater than or equal to about 10⁷M⁻¹, more preferably greater than or equal to about 10⁸ M⁻¹, and mostpreferably greater than or equal to about 10⁹ M⁻¹, 10¹⁹ M⁻¹, 10¹¹ M⁻or10¹² M⁻¹. The anti-OSM antibodies may bind to different naturallyoccurring forms of OSM, including those expressed by thehost's/subject's tissues as well as that expressed by the tumor. Themonoclonal antibodies disclosed herein, such as RX1, 5H4, MC1, or MC3antibody, have affinity for OSM and are characterized by a dissociationequilibrium constant (K^(d)) of at least 10⁻⁴ M, preferably at leastabout 10⁻⁷ M to about 10⁻⁸ M, more preferably at least about 108 M,10^(10M), 10^(−11M) or 10^(−12M). Such affinities may be readilydetermined using conventional techniques, such as by equilibriumdialysis; by using the BIAcore 2000 instrument, using general proceduresoutlined by the manufacturer; by radioimmunoassay using ¹²⁵I labeledOSM; or by another method known to the skilled artisan. The affinitydata may be analyzed, for example, by the method of Scatchard et al.,Ann N.Y. Acad. Sci., 51:660 (1949). Thus, it will be apparent thatpreferred OSM antibodies will exhibit a high degree of specificity forOSM and will bind with substantially lower affinity to other molecules.

The antigen to be used for production of antibodies may be, e.g., intactOSM or a fragment of OSM that retains the desired epitope, optionallyfused to another polypeptide that allows the epitope to be displayed inits native conformation. Alternatively, cells expressing OSM at theircell surface can be used to generate antibodies. Such cells can betransformed to express OSM or may be other naturally occurring cellsthat express OSM. Other forms of OSM useful for generating antibodieswill be apparent to those skilled in the art.

Anti-OSM antibodies are known in the art and disclosed in US20130251724and US20140099315, the disclosures of which are incorporated herein byreference.

C. OSM Muteins

The invention further provides OSM muteins that may be used as OSMantagonists according to the methods of the invention.

“Fragment” as used herein means a portion of the intact native molecule;for example, a fragment polypeptide is a fragment of the nativepolypeptide in which one or more amino acids from either the N-terminalor C-terminal have been deleted.

“Mutein” as used herein with respect to polypeptides means a variant ofthe intact native molecule or a variant of a fragment of the nativemolecule, in which one or more amino acids have been substituted,inserted or deleted. Such substitutions, insertions or deletions can beat the N-terminus, C-terminus or internal to the molecule. Thus the term“muteins” includes within its scope fragments of the native molecule.Insertional muteins include fusions at the N- or C-terminus, e.g. fusionto the Fc portion of an immunoglobulin to increase half-life.

Preferred muteins according to the invention exhibit at least about 65%,70%. 75%, 80%, 85%, 90%, 95%, 97% or more sequence identity (homology)to the native polypeptide, as determined by the Smith-Waterman homologysearch algorithm (Meth. Mol. Biol. 70:173-187 (1997)) as implemented inthe MSPRCH program (Oxford

Molecular) using an affine gap search with the following searchparameters: gap open penalty of 12, and gap extension penalty of 1.Other well-known and routinely used homology/identity scanning algorithmprograms include Pearson and Lipman, PNAS USA, 85:2444-2448 (1988);Lipman and Pearson, Science, 222:1435 (1985); Devereaux et al., Nuc.Acids Res., 12:387-395 (1984); or the BLASTP, BLASTN or BLASTXalgorithms of Altschul, et al., Mol. Biol., 215:403-410 (1990).Computerized programs using these algorithms are also available andinclude, but are not limited to: GAP, BESTFIT, BLAST, FASTA and TFASTA,which are commercially available from the Genetics Computing Group (GCG)package, Version 8, Madison Wis., USA; and CLUSTAL in the PC/Geneprogram by Intellegenetics, Mountain View Calif. Preferably, thepercentage of sequence identity is determined by using the defaultparameters determined by the program.

“Modification” as used herein means any modification of the nativepolypeptide, fragment or mutein, such as glycosylation, phosphorylation,polymer conjugation (such as with polyethylene glycol), or otheraddition of foreign moieties, so long as the desired activity (agonistor antagonist) is retained.

In yet another embodiment, the OSM mutein comprises one or more ofbinding sites 1, 2, or 3, or portions thereof involved inreceptor-binding, alone or fused to other polypeptides that allowdisplay of the fragments in proper three-dimensional conformation.

Muteins containing any desired conservative and/or non-conservativemuteins are readily prepared using techniques well known in the art,including recombinant production or chemical synthesis.

Conservative substitutions, particularly substitutions outside ofregions directly involved in ligand-receptor binding, are not expectedto significantly change the binding properties of the OSM muteins (orOSMR muteins). Amino acids can be classified according to physicalproperties and contribution to secondary and tertiary protein structure.A conservative substitution is recognized in the art as a substitutionof one amino acid for another amino acid that has similar properties.Exemplary conservative substitutions are set out in Table 2 (from WO97/09433, page 10, published Mar. 13, 1997 (PCT/GB96/02197, filed Sep.6, 1996), immediately below.

The availability of a DNA sequence encoding OSM permits the use ofvarious expression systems to produce the desired polypeptides.Construction of expression vectors and recombinant production from theappropriate DNA sequences are performed by methods well known in theart. These techniques and various other techniques are generallyperformed according to Sambrook et al., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989),and Kriegler, M., Gene Transfer and Expression, A Laboratory Manual,Stockton Press, New York (1990), both of which are incorporated hereinby reference.

Certain modifications to the primary sequence of OSM can be made bydeletion, addition, or alteration of the amino acids encoded by the DNAsequence without destroying the desired structure (e.g., the receptorbinding ability of OSM) in accordance with well-known recombinant DNAtechniques. Further, a skilled artisan will appreciate that individualamino acids may be substituted or modified by oxidation, reduction orother modification, and the polypeptide may be cleaved to obtainfragments that retain the active binding site and structuralinformation. Such substitutions and alterations result in polypeptideshaving an amino acid sequence which falls within the definition ofpolypeptide “having substantially the same amino acid sequence” as themature OSM SEQ ID NO:1 and 2.

Polypeptides may be produced by chemical synthesis or recombinantproduction techniques known in the art.

The relatedness of proteins can also be characterized through therelatedness of their encoding nucleic acids. Methods to determineidentity and/or similarity of polynucleotide sequences are describedabove. In addition, methods to determine similarity of polynucleotidesequences through testing their ability to hybridize under moderately orhighly stringent conditions may be determined as follows. Exemplarymoderately stringent hybridization conditions are as follows:hybridization at 42° C. in a hybridization solution comprising 50%formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate, and washing twice for30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS.Highly stringent conditions include washes at 68° C. in a wash solutioncomprising 0.1×SSC and 1% SDS. It is understood in the art thatconditions of equivalent stringency can be achieved through variation oftemperature and buffer, or salt concentration as described in the art(Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley &Sons (1994), pp. 6.0.3 to 6.4.10). Modifications in hybridizationconditions can be empirically determined or precisely calculated basedon the length and the percentage of guanosine/cytosine (GC) base pairingof the probe. The hybridization conditions can be calculated asdescribed in Sambrook et al., (Eds.), Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.(1989), pp. 9.47 to 9.51.

D. OSM Gene Therapy

Delivery of a therapeutic protein to appropriate cells can be effectedvia gene therapy ex vivo, in situ, or in vivo by use of any suitableapproach known in the art, including by use of physical DNA transfermethods (e.g., liposomes or chemical treatments) or by use of viralvectors (e.g., adenovirus, adeno-associated virus, or a retrovirus).Antisense compounds and methods of using them are also provided by thepresent invention. The level of OSM or OSMR activity may be reduced byusing well-known antisense, gene “knock-out,” ribozyme, triple helix orRNAi methods to decrease the level gene expression. Techniques for theproduction and use of such molecules are well known to those of skill inthe art.

As used herein, the term “peptidomimetic” is a non-peptide compound thatcomprises an assembly of amino acid side chains, or pharmacophores, orsuitable derivatives thereof, that are supported on a scaffold such thatthe spatial orientation of the pharmacophores substantially mimic thebioactive conformation of a natural peptide. For example, apeptidomimetic may lack amino acids or peptide bonds but retain theparticular three-dimensional arrangement of peptide chain groups fromthe parent peptide that is required for binding activity. The scaffoldmay comprise a bicyclic, tricyclic or higher polycyclic carbon orheteroatom skeleton, or may be based on one or more ring structures(e.g., pyridine, imidazole, etc.) or amide bonds. This scaffold may belinked by spacers to an acidic group (e.g. a carboxylic acid functionalgroup) at one end and a basic group (e.g. an N-containing moiety such asamidine or guanidine) at the other end of the core. Exemplary techniquesfor synthesizing peptidomimetics are described in U.S. patentapplication no. 20030199531 published Oct. 23, 2003, U.S. PatentApplication No. 20030139348 published Jul. 24, 2003.

In addition to antibodies and other proteins, this invention alsocontemplates alternative OSM antagonists including, but not limited to,peptides or small organic molecules that are also effective ininhibiting the interaction between OSM and OSMR or the activation ofOSMR.

II.) Combination Therapy

Concurrent administration of two therapeutic agents according to thepresent invention, such as an OSM antagonist and a secondanti-osteoclast agent, does not require that the agents be administeredat the same time or by the same route, as long as there is an overlap inthe time period during which the agents are exerting their therapeuticeffect. Simultaneous or sequential administration is contemplated, as isadministration on different days or weeks.

The discovery of a significant time lag to observe therapeutic effectafter commencing treatment with an OSM antibody (an exemplary OSMantagonist) makes desirable the co-administration of a secondanti-osteoclast agent with quicker onset of action during thistransition period. During the transition period, the two agents must beadministered at a monotherapeutically effective amount. Subsequent tothe transition period, the second anti-osteoclast agent may bediscontinued or reduced in dosage. If the OSM antagonist and secondanti-osteoclast agent exert synergistic effects, the dose of one or bothmay be lowered after the transition period.

Compositions of the invention are administered to a mammal alreadysuffering from, or predisposed to, cancer and associated tumor celldetachment, proliferation and/or metastasis, in an amount sufficient toprevent or at least partially arrest the development of such disease. Anamount of a therapeutic agent adequate to accomplish this when thetherapeutic agent is given alone (not in combination with a secondtherapeutic agent) is defined as a “monotherapeutically effective dose.”

In the combination therapy methods of the present invention, the OSMantagonist, such as the OSM antibody, and the second anti-osteoclastagent may be administered simultaneously or at different time. The twoagents can be administered, for example, within 8 hours, 1 day, 14 days,30 days, 3 months, 6 months, 9 months or 1 year of each other.

Exemplary second anti-osteoclast agents include bisphosphonates,including but not limited to zoledronate, pamidronate, clodronate,etidronate, tiludronate, alendronate, ibandronate or risedronate.Exemplary other anti-osteoclast agents include bisphosphonates, PTHrPneutralizing agents (e.g., antibody, antisense, siRNA), cathepsin Kinhibitors, MIP-1-{acute over (α)} antagonists, RANK/RANKL neutralizingagents (e.g., anti-RANK antibody, such as AMG-162, or antisense, solubleRANKL receptor or muteins thereof), RANKL vaccine, osteoprotegrin (OPG),platelet-derived growth factors (PDGF), src kinase inhibitors, galliummaltolate, and matrix metalloproteinase (MMP) inhibitors.

Exemplary doses of bisphosphonates include the intravenousadministration of 4 mg. Lesser dosages may also be administeredincluding 3.5 mg, 3.3 mg or 3.0 mg. Other routes of administration arepossible including subcutaneous and as described in WO 02/087555.Effective amounts of a OSM antibody will vary and depend on the severityof the disease and the weight and general state of the patient beingtreated, but generally range from about 1.0 mg/kg to about 100 mg/kgbody weight, or about 10 mg/kg to about 30 mg/kg, with dosages of fromabout 0.1 mg/kg to about 10 mg/kg or about 1 mg/kg to about 10 mg/kg perapplication being more commonly used. For example, about 10 mg/kg to 5mg/kg or about 30 mg/kg to 1 mg/kg of antibody is an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion.Administration is daily, on alternating days, weekly or less frequently,as necessary depending on the response to the disease and the patient'stolerance of the therapy. Maintenance dosages over a longer period oftime, such as 4, 5, 6, 7, 8, 10 or 12 weeks or longer may be neededuntil a desired suppression of disease symptoms occurs, and dosages maybe adjusted as necessary. The progress of this therapy is easilymonitored by conventional techniques and assays.

Although the methods of the present invention may be useful for allstages of cancers, they may be particularly appropriate in advanced ormetastatic cancers. Combining the therapy method with a chemotherapeuticor radiation regimen may be preferred in patients that have not receivedchemotherapeutic treatment, whereas treatment with the therapy method ofthe present invention may be indicated for patients who have receivedone or more chemotherapies. Additionally, the therapy methods of thepresent invention can also enable the use of reduced dosages ofconcomitant chemotherapy, particularly in patients that do not toleratethe toxicity of the chemotherapeutic agent very well.

The method of the invention contemplates the administration of singleanti-OSM antibodies, as well as combinations, or “cocktails”, ofdifferent antibodies. Such antibody cocktails may have certainadvantages in as much as they contain antibodies which exploit differenteffector mechanisms or combine directly cytotoxic antibodies withantibodies that rely on immune effector functionality. Such antibodiesin combination may exhibit synergistic therapeutic effects.

The methods of the invention can be used in combination with yet othertherapeutics, such as cancer therapeutics. Exemplary cancer therapeuticagents and/or procedures, include but are not limited to variouschemotherapeutic agents, androgen-blockers, and immune modulators (e.g.,IL-2, GOSM, SLC), Bisphosphonate(s) (e.g.,

Aredia (i.e., pamidronate, pamidronic acid, disodium pamidronate,pamidronate disodium pentahydrate); Zometa (i.e., Aclasta, zoledronicacid, zoledronate); Clondronate (i.e., Bonefos, Loron, clodronatedisodium, sodium clondronate); Fosamax (i.e., alendronate, alendronatesodium salt trihydrate, alendronic acid); Fosavance (i.e., Fosamaxformulated with vitamin D); Bondronat or Bonviva or Boniva (i.e.,ibandronate, ibandronic acid, ibandronate sodium); Actonel (i.e.,risedronate, risedronate sodium, risendronic acid); Didronel or Didrocal(i.e., etidronate, etidronic acid, etidronate disodium); Nerixia (i.e.,neridronate, neridronic acid); Skelid (i.e., tiludronate, tiludronicacid); dimethyl-APD (i.e., olpadronate, olpadronic acid); and medronicacid or medronate), surgery, radiation, cytotoxic chemotherapy, hormonetherapy (e.g., Tamoxifen; anti-Androgen therapy), antibody therapy(e.g., antibodies to RANKL/RANK neutralizing; PTHrP neutralizing,anti-Her2, anti-CD20, anti-CD40, CD22, VEGF, IGFR-1, EphA2, HAAH,TMEFF2, CAIX antibodies), therapeutic protein therapy (e.g., solubleRANKL receptor; OPG, and PDGF and MMP inhibitors), small molecule drugtherapy (e.g., Src-kinase inhibitor), kinase inhibitors of growth factorreceptors, or RANKL inhibitors, oligonucleotides therapy (e.g., RANKL orRANK or PTHrP Anti-sense), gene therapy (e.g. RANKL or RANK inhibitors,such as anti-RANKL antibodies), peptide therapy (e.g. muteins of RANKL)as well as those proteins, peptides, compounds, and small moleculesdescribed herein.

Cancer chemotherapeutic agents include, without limitation, alkylatingagents, such as carboplatin and cisplatin; nitrogen mustard alkylatingagents; nitrosourea alkylating agents, such as carmustine (BCNU);antimetabolites, such as methotrexate; folinic acid;

purine analog antimetabolites, mercaptopurine; pyrimidine analogantimetabolites, such as fluorouracil (5-FU) and gemcitabine (Gemzar®.);hormonal antineoplastics, such as goserelin, leuprolide, and tamoxifen;natural antineoplastics, such as aldesleukin, interleukin-2, docetaxel,eloposide (VP-16), interferon alfa, paclitaxel (Taxol®), and tretinoin(ATRA); antibiotic natural antineoplastics, such as bleomycin,dactinomycin, daunorubicin, doxorubicin, daunomycin and mitomycinsincluding mitomycin C; and vinca alkaloid natural antineoplastics, suchas vinblastine, vincristine, vindesine; hydroxyurea; aceglatone,adriamycin, ifosfamide, enocitabine, epitiostanol, aclarubicin,ancitabine, nimustine, procarbazine hydrochloride, carboquone,carboplatin, carmofur, chromomycin A3, antitumor polysaccharides,antitumor platelet factors, cyclophosphamide (Cytoxin®); Schizophyllan,cytarabine® (cytosine arabinoside), dacarbazine, thioinosine, thiotepa,tegafur, dolastatins, dolastatin analogs such as auristatin, CPT-11(irinotecan), mitozantrone, vinorelbine, teniposide, aminopterin,caminomycin, esperamicins (See, e.g., U.S. Pat. No. 4,675,187),neocarzinostatin, OK-432, bleomycin, furtulon, broxuridine, busulfan,honvan, peplomycin, bestatin (Ubenimex®.), interferon-β, mepitiostane,mitobronitol, melphalan, laminin peptides, lentinan, Coriolus versicolorextract, tegafur/uracil, estramustine (estrogen/mechlorethamine).

Further, additional agents used as therapy for cancer patients includeEPO, G-CSF, ganciclovir; antibiotics, leuprolide; meperidine; zidovudine(AZT); interleukins 1 through 18, including mutants and analogues;interferons or cytokines, such as interferons α, β, and γ hormones, suchas luteinizing hormone releasing hormone (LHRH) and analogues and,gonadotropin releasing hormone (GnRH); growth factors, such astransforming growth factor-β (TGF-(β), fibroblast growth factor (FGF),nerve growth factor (NGF), growth hormone releasing factor (GHRF),epidermal growth factor (EGF), fibroblast growth factor homologousfactor (FGFHF), hepatocyte growth factor (HGF), and insulin growthfactor (IGF); tumor necrosis factor-α & β (TNF-α & β); invasioninhibiting factor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7);somatostatin; thymosin-α-1; γ-globulin; superoxide dismutase (SOD);complement factors; anti-angiogenesis factors: antigenic materials; andpro-drugs.

Prodrug refers to a precursor or derivative form of a pharmaceuticallyactive substance that is less cytotoxic or non-cytotoxic to tumor cellscompared to the parent drug and is capable of being enzymaticallyactivated or converted into an active or the more active parent form.See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical SocietyTransactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stellaet al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,”Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, HumanaPress (1985). Prodrugs include, but are not limited to,phosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate-containing prodrugs, peptide-containing prodrugs, D-aminoacid-modified prodrugs, glycosylated prodrugs, β-lactam-containingprodrugs, optionally substituted phenoxyacetamide-containing prodrugs oroptionally substituted phenylacetamide-containing prodrugs,5-fluorocytosine and other 5-fluorouridine prodrugs which can beconverted into the more active cytotoxic free drug. Examples ofcytotoxic drugs that can be derivatized into a prodrug form for useherein include, but are not limited to, those chemotherapeutic agentsdescribed above.

III). Compositions Comprising OSM Antagonists

Compositions for inhibiting or reducing tumor cell detachment,proliferation and/or metastasis are provided. Preferred compositions arethose that interfere, inhibit, reduce or block OSM protein function. Inone embodiment the composition includes an antagonist of OSM, or itstarget gp130 or a combination thereof. A preferred OSM proteinantagonist includes, but is not limited to a small molecule thatsterically interacts with binding site 1 of OSM.

Other embodiments provide compositions for inhibiting or reducing OSMactivity, such as antibodies which bind and thus inhibit OSM activity,proteins, muteins, and/or nucleic acid compositions which may interferewith OSM production or may encode antibodies and other proteininhibitors themselves.

Another embodiment is directed to compositions comprising an OSMantagonist in an amount effective to inhibit OSM activity relative to acontrol. It will be appreciated that a control includes cells ororganisms that are not treated with the disclosed compositions.

In another embodiment, the disclosed OSM protein antagonists selectivelyinteract with a region disclosed herein as active site 1 of the OSMprotein.

The compositions are administered to an individual in need of treatmentor prophylaxis of at least one symptom or manifestation (since diseasecan occur/progress in the absence of symptoms) of cancer or cellularhyperproliferation. In one embodiment, the compositions are administeredin an effective amount to inhibit OSM mediated cellular activity andthereby inhibit or reduce tumor cell detachment, proliferation and/ormetastasis. The amount of inhibition can be determined relative to acontrol, for example cells that are not treated with the inhibitor.Methods for measuring inhibition OSM activity are provided in theExamples.

A. Formulations

The compounds are preferably employed for therapeutic uses incombination with a suitable pharmaceutical carrier. Such compositionsinclude an effective amount of the compound, and a pharmaceuticallyacceptable carrier or excipient. The formulation is made to suit themode of administration. Pharmaceutically acceptable carriers aredetermined in part by the particular composition being administered, aswell as by the particular method used to administer the composition.Accordingly, there is a wide variety of suitable formulations ofpharmaceutical compositions containing the nucleic acids some of whichare described herein.

The compounds may be in a formulation for administration topically,locally or systemically in a suitable pharmaceutical carrier.Remington's Pharmaceutical Sciences, 15th Edition by E. W. Martin (MarkPublishing Company, 1975), discloses typical carriers and methods ofpreparation. The compound may also be encapsulated in suitablebiocompatible microcapsules, microparticles or microspheres formed ofbiodegradable or non-biodegradable polymers or proteins or liposomes fortargeting to cells. Such systems are well known to those skilled in theart and may be optimized for use with the appropriate nucleic acid.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases, orthickeners can be used as desired.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions, solutions or emulsions thatcan include suspending agents, solubilizers, thickening agents,dispersing agents, stabilizers, and preservatives. Formulations forinjection may be presented in unit dosage form, e.g., in ampules or inmulti-dose containers, with an added preservative.

Preparations include sterile aqueous or nonaqueous solutions,suspensions and emulsions, which can be isotonic with the blood of thesubject in certain embodiments.

Examples of nonaqueous solvents are polypropylene glycol, polyethyleneglycol, vegetable oil such as olive oil, sesame oil, coconut oil,arachis oil, peanut oil, mineral oil, injectable organic esters such asethyl oleate, or fixed oils including synthetic mono or di-glycerides.Aqueous carriers include water, alcoholic/aqueous solutions, emulsionsor suspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, 1,3-butandiol, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, antioxidants, chelating agents and inertgases and the like. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose any blandfixed oil may be employed including synthetic mono- or di-glycerides. Inaddition, fatty acids such as oleic acid may be used in the preparationof injectables. Carrier formulation can be found in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa. Those of skillin the art can readily determine the various parameters for preparingand formulating the compositions without resort to undueexperimentation.

The compound alone or in combination with other suitable components, canalso be made into aerosol formulations (i.e., they can be “nebulized”)to be administered via inhalation. Aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. Foradministration by inhalation, the compounds are conveniently deliveredin the form of an aerosol spray presentation from pressurized packs or anebulizer, with the use of a suitable propellant.

In some embodiments, the compound described above may includepharmaceutically acceptable carriers with formulation ingredients suchas salts, carriers, buffering agents, emulsifiers, diluents, excipients,chelating agents, fillers, drying agents, antioxidants, antimicrobials,preservatives, binding agents, bulking agents, silicas, solubilizers, orstabilizers. In one embodiment, the compounds are conjugated tolipophilic groups like cholesterol and lauric and lithocholic acidderivatives with C32 functionality to improve cellular uptake. Forexample, cholesterol has been demonstrated to enhance uptake and serumstability of siRNA in vitro (Lorenz, et al., Bioorg. Med. Chem. Lett.14(19):4975-4977 (2004)) and in viva (Soutschek, et al., Nature432(7014):173-178 (2004)). Other groups that can be attached orconjugated to the compounds described above to increase cellular uptake,include acridine derivatives; cross-linkers such as psoralenderivatives, azidophenacyl, proflavin, and azidoproflavin; artificialendonucleases; metal complexes such as EDTA-Fe(II) and porphyrin-Fe(II);alkylating moieties; enzymes such as alkaline phosphatase; terminaltransferases; abzymes; cholesteryl moieties; lipophilic carriers;peptide conjugates; long chain alcohols; phosphate esters; radioactivemarkers; non-radioactive markers; carbohydrates; and polylysine or otherpolyamines. U.S. Pat. No. 6,919,208 to Levy, et al., also describedmethods for enhanced delivery. These pharmaceutical formulations may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

B. Methods of Administration

In general, methods of administering compounds are well known in theart. The compositions can be administered by a number of routesincluding, but not limited to: oral, intravenous, intraperitoneal,intramuscular, transdermal, subcutaneous, topical, sublingual, or rectalmeans. Compounds can also be administered via liposomes. Suchadministration routes and appropriate formulations are generally knownto those of skill in the art.

Administration of the formulations described herein may be accomplishedby any acceptable method which allows the compounds to reach its target.The particular mode selected will depend of course, upon factors such asthe particular formulation, the severity of the state of the subjectbeing treated, and the dosage required for therapeutic efficacy. Asgenerally used herein, an “effective amount” is that amount which isable to treat one or more symptoms of age related disorder, reverse theprogression of one or more symptoms of age related disorder, halt theprogression of one or more symptoms of age related disorder, or preventthe occurrence of one or more symptoms of age related disorder in asubject to whom the formulation is administered, as compared to amatched subject not receiving the compound. The actual effective amountsof compound can vary according to the specific compound or combinationthereof being utilized, the particular composition formulated, the modeof administration, and the age, weight, condition of the individual, andseverity of the symptoms or condition being treated.

Any acceptable method known to one of ordinary skill in the art may beused to administer a formulation to the subject. The administration maybe localized (i.e., to a particular region, physiological system,tissue, organ, or cell type) or systemic, depending on the conditionbeing treated.

Injections can be e.g., intravenous, intradermal, subcutaneous,intramuscular, or intraperitoneal. The composition can be injectedintradermally for treatment or prevention of age related disorder, forexample. In some embodiments, the injections can be given at multiplelocations. Implantation includes inserting implantable drug deliverysystems, e.g., microspheres, hydrogels, polymeric reservoirs,cholesterol matrixes, polymeric systems, e.g., matrix erosion and/ordiffusion systems and non-polymeric systems, e.g., compressed, fused, orpartially-fused pellets. Inhalation includes administering thecomposition with an aerosol in an inhaler, either alone or attached to acarrier that can be absorbed. For systemic administration, it may bepreferred that the composition is encapsulated in liposomes.

The formulations may be delivered using a bioerodible implant by way ofdiffusion or by degradation of the polymeric matrix. In certainembodiments, the administration of the formulation may be designed so asto result in sequential exposures to the active agent over a certaintime period, for example, hours, days, weeks, months or years. This maybe accomplished, for example, by repeated administrations of aformulation or by a sustained or controlled release delivery system inwhich the active agent is delivered over a prolonged period withoutrepeated administrations. Administration of the formulations using sucha delivery system may be, for example, by oral dosage forms, bolusinjections, transdermal patches or subcutaneous implants. Maintaining asubstantially constant concentration of the composition may be preferredin some cases.

Other delivery systems suitable include time-release, delayed release,sustained release, or controlled release delivery systems. Such systemsmay avoid repeated administrations in many cases, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude, for example, polymer-based systems such as polylactic and/orpolyglycolic acids, polyanhydrides, polycaprolactones, copolyoxalates,polyesteramides, polyorthoesters, polyhydroxybutyric acid, and/orcombinations of these. Microcapsules of the foregoing polymerscontaining nucleic acids are described in, for example, U.S. Pat. No.5,075,109. Other examples include nonpolymer systems that arelipid-based including sterols such as cholesterol, cholesterol esters,and fatty acids or neutral fats such as mono-, di- and triglycerides;hydrogel release systems; liposome-based systems; phospholipidbased-systems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; orpartially fused implants. Specific examples include erosional systems inwhich the OSM protein antagonist is contained in a formulation within amatrix (for example, as described in U.S. Pat. Nos. 4,452,775,4,675,189, 5,736,152, 4,667,013, 4,748,034 and 5,239,660), ordiffusional systems in which an active component controls the releaserate (for example, as described in U.S. Pat. Nos. 3,832,253, 3,854,480,5,133,974 and 5,407,686). The formulation may be as, for example,microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, orpolymeric systems. In some embodiments, the system may allow sustainedor controlled release of the composition to occur, for example, throughcontrol of the diffusion or erosion/degradation rate of the formulationcontaining the OSM protein antagonist. In addition, a pump-basedhardware delivery system may be used to deliver one or more embodiments.

Examples of systems in which release occurs in bursts includes, e.g.,systems in which the composition is entrapped in liposomes which areencapsulated in a polymer matrix, the liposomes being sensitive tospecific stimuli, e.g., temperature, pH, light or a degrading enzyme andsystems in which the composition is encapsulated by an ionically-coatedmicrocapsule with a microcapsule core degrading enzyme. Examples ofsystems in which release of the inhibitor is gradual and continuousinclude, e.g., erosional systems in which the composition is containedin a form within a matrix and effusional systems in which thecomposition permeates at a controlled rate, e.g., through a polymer.Such sustained release systems can be e.g., in the form of pellets, orcapsules.

Use of a long-term release implant may be particularly suitable in someembodiments. “Long-term release,” as used herein, means that the implantcontaining the composition is constructed and arranged to delivertherapeutically effective levels of the composition for at least 30 or45 days, and preferably at least 60 or 90 days, or even longer in somecases. Long-term release implants are well known to those of ordinaryskill in the art, and include some of the release systems describedabove.

C. Effective Dosages

Dosages for a particular individual can be determined by one of ordinaryskill in the art using conventional considerations, (e.g. by means of anappropriate, conventional pharmacological protocol). A physician may,for example, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. The doseadministered to an individual is sufficient to effect a beneficialtherapeutic response in the individual over time, or, e.g., to reducesymptoms, or other appropriate activity, depending on the application.The dose is determined by the efficacy of the particular formulation,and the activity, stability or serum half-life of the OSM proteinantagonist employed and the condition of the individual, as well as thebody weight or surface area of the individual to be treated. The size ofthe dose is also determined by the existence, nature, and extent of anyadverse side-effects that accompany the administration of a particularvector, formulation, or the like in a particular individual.

Formulations are administered at a rate determined by the LD50 of therelevant formulation, and/or observation of any side-effects of thecompositions at various concentrations, e.g., as applied to the mass andoverall health of the individual. Administration can be accomplished viasingle or divided doses.

In vitro models can be used to determine the effective doses of thecompositions as a potential cancer treatment, as described in theexamples. In determining the effective amount of the compound to beadministered in the treatment or prophylaxis of disease the physicianevaluates circulating plasma levels, formulation toxicities, andprogression of the disease. For the disclosed compositions, the doseadministered to a 70 kilogram individual is typically in the rangeequivalent to dosages of currently-used therapeutic antibodies such asAvastin®, Erbitux® and Herceptin®.

The formulations described herein can supplement treatment conditions byany known conventional therapy, including, but not limited to, antibodyadministration, vaccine administration, administration of cytotoxicagents, natural amino acid polypeptides, nucleic acids, nucleotideanalogues, and biologic response modifiers. Two or more combinedcompounds may be used together or sequentially. For example, thecompositions can also be administered in therapeutically effectiveamounts as a portion of an anti-cancer cocktail. Anti-cancer cocktailscan include therapeutics to treat cancer or angiogenesis of tumors.

IV). Methods of Treatment

The disclosed compositions can be administered to a subject in needthereof to treat, alleviate, or reduce one or more symptoms associatedwith cancer or other forms of cellular hyperproliferation. Thecompositions can be administered locally or systemically to inhibittumor cell detachment, proliferation and/or metastasis. The types ofcancer that can be treated with the provided compositions and methodsinclude, but are not limited to, the following: bladder, brain, breast,cervical, colorectal, esophageal, kidney, liver, lung, nasopharangeal,pancreatic, prostate, skin, stomach, uterine, ovarian, and testicular.In a preferred embodiment the cancer is prostate or breast cancer.Administration is not limited to the treatment of an existing tumor butcan also be used to prevent or lower the risk of developing suchdiseases in an individual, i.e., for prophylactic use. Potentialcandidates for treatment include individuals with a high risk ofdeveloping cancer, i.e., with a personal or familial history of certaintypes of cancer.

Malignant tumors which may be treated are classified herein according tothe embryonic origin of the tissue from which the tumor is derived.Carcinomas are tumors arising from endodermal or ectodermal tissues suchas skin or the epithelial lining of internal organs and glands.Sarcomas, which arise less frequently, are derived from mesodermalconnective tissues such as bone, fat, and cartilage. The leukemias andlymphomas are malignant tumors of hematopoietic cells of the bonemarrow. Leukemias proliferate as single cells, whereas lymphomas tend togrow as tumor masses. Malignant tumors may show up at numerous organs ortissues of the body to establish a cancer.

In one embodiment, the subject is subjected to primary surgery relatedto the cancer. In another embodiment, administering the pharmaceuticalformulation takes place before, during or after the primary surgery. Inyet another embodiment, the cancer comprises a solid cancer. In yetanother embodiment, the solid cancer is selected from the groupconsisting of breast cancer and prostate cancer.

V). Methods for Screening for Inhibitors of Tumor Cell Detachment,Proliferation and Metastasis

Methods for identifying inhibitors of tumor cell detachment,proliferation and/or metastasis are provided and utilize well knowntechniques and reagents. The inhibitor reduces, inhibits, blocks, orinterferes with OSM protein function, expression, or bioavailability.

In some embodiments, the assays can include random screening of largelibraries of test compounds. The test compounds are, in a preferredembodiment non-protein small molecules. The term “small molecule” refersto compounds less than 1,000 daltons, typically less than 500 daltons.Alternatively, the assays may be used to focus on particular classes ofcompounds suspected of inhibiting OSM activity in cells, tissues,organs, or systems.

Assays can include determinations of OSM protein expression, proteinexpression, protein activity, signal transduction, or binding activity.Other assays can include determinations of OSM protein nucleic acidtranscription or translation, for example mRNA levels, mRNA stability,mRNA degradation, transcription rates, and translation rates.

In one embodiment, the identification of an inhibitor of tumor celldetachment, proliferation and/or metastasis is based on the function ofOSM in the presence and absence of a test compound. The test compound ormodulator can be any substance that alters or is believed to alter thefunction of OSM. Typically, an inhibitor will be selected that reduces,eliminates, or inhibits OSM and the OSM initiated regulatory pathway.

One exemplary method includes contacting OSM protein with at least afirst test compound, and assaying for an interaction between OSM proteinand the first test compound with an assay. The assaying can includedetermining inhibition of OSM interaction with gp-130.

Specific assay endpoints or interactions that may be measured in thedisclosed embodiments include assaying for OSM, modulation, down or upregulation or turnover. These assay endpoints may be assayed usingstandard methods such as FACS, FACE, ELISA, Northern blotting and/orWestern blotting. Moreover, the assays can be conducted in cell freesystems, in isolated cells, genetically engineered cells, immortalizedcells, or in organisms such as transgenic animals.

Other screening methods include using labeled OSM protein to identify atest compound. OSM can be labeled using standard labeling proceduresthat are well known and used in the art. Such labels include, but arenot limited to, radioactive, fluorescent, biological and enzymatic tags.

Another embodiment provides a method for identifying an inhibitor oftumor cell detachment, proliferation and/or metastasis by determiningthe effect a test compound has OSM activity. For example isolated cellsor whole organisms expressing OSM or both can be contacted with a testcompound. OSM activity can be determined using standard biochemicaltechniques such as immunodetection. Suitable cells for this assayinclude, but are not limited to, cancer cells, immortalized cell lines,primary cell culture, or cells engineered to express OSM proteins, forexample cells from mammals such as humans. Compounds that inhibit OSMactivity can be selected.

Another embodiment provides for in vitro assays for the identificationof inhibitors of tumor cell detachment, proliferation and/or metastasis.Such assays generally use isolated molecules, can be run quickly and inlarge numbers, thereby increasing the amount of information obtainablein a short period of time. A variety of vessels may be used to run theassays, including test tubes, plates, dishes and other surfaces such asdipsticks or beads.

One example of a cell free assay is a binding assay. While not directlyaddressing function, the ability of a modulator to bind to a targetmolecule in a specific fashion is strong evidence of a relatedbiological effect. Such a molecule can bind to OSM protein and inhibitits biological functions. The binding of a molecule to a target may, inand of itself, be inhibitory, due to steric, allosteric or charge—chargeinteractions or inactivation of OSM protein. The target may be eitherfree in solution, fixed to a support, expressed in or on the surface ofa cell. Either the target or the compound may be labeled, therebypermitting determining of binding. Usually, the target will be thelabeled species, decreasing the chance that the labeling will interferewith or enhance binding. Competitive binding formats can be performed inwhich one of the agents is labeled, and one may measure the amount offree label versus bound label to determine the effect on binding.

A technique for high throughput screening of compounds is described inWO 84/03564. Large numbers of small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. Bound polypeptide is detected by various methods.

Other embodiments include methods of screening compounds for theirability to inhibit the function of OSM proteins. Various cell lines canbe utilized for such screening assays, including cells specificallyengineered for this purpose. Furthermore, those of skill in the art willappreciate that stable or transient transfections, which are well knownand used in the art, may be used in the disclosed embodiments.

For example, a transgenic cell comprising an expression vector can begenerated by introducing the expression vector into the cell. Theintroduction of DNA into a cell or a host cell is well known technologyin the field of molecular biology and is described, for example, inSambrook et al., Molecular Cloning 3rd Ed. (2001). Methods oftransfection of cells include calcium phosphate precipitation, liposomemediated transfection, DEAE dextran mediated transfection,electroporation, ballistic bombardment, and the like. Alternatively,cells may be simply transfected with the disclosed expression vectorusing conventional technology described in the references and examplesprovided herein. The host cell can be a prokaryotic or eukaryotic cell,or any transformable organism that is capable of replicating a vectorand/or expressing a heterologous gene encoded by the vector. Numerouscell lines and cultures are available for use as a host cell, and theycan be obtained through the American Type Culture Collection (ATCC),which is an organization that serves as an archive for living culturesand genetic materials (www.atcc.org).

A host cell can be selected depending on the nature of the transfectionvector and the purpose of the transfection. A plasmid or cosmid, forexample, can be introduced into a prokaryote host cell for replicationof many vectors. Bacterial cells used as host cells for vectorreplication and/or expression include DH5.alpha., JM109, and KCB, aswell as a number of commercially available bacterial hosts such as SURE®Competent Cells and SOLOPACK Gold Cells (STRATAGENE, La Jolla, Calif.).Alternatively, bacterial cells such as E. coli LE392 could be used ashost cells for phage viruses. Eukaryotic cells that can be used as hostcells include, but are not limited to, yeast, insects and mammals.

Examples of mammalian eukaryotic host cells for replication and/orexpression of a vector include, but are not limited to, HeLa, NIH3T3,Jurkat, 293, Cos, CHO, Saos, and PC12. Examples of yeast strains includeYPH499, YPH500 and YPH501. Many host cells from various cell types andorganisms are available and would be known to one of skill in the art.Similarly, a viral vector may be used in conjunction with either aneukaryotic or prokaryotic host cell, particularly one that is permissivefor replication or expression of the vector. Depending on the assay,culture may be required. The cell is examined using any of a number ofdifferent physiologic assays. Alternatively, molecular analysis may beperformed, for example, looking at protein expression, mRNA expression(including differential display of whole cell or polyA RNA) and others.

In vivo assays involve the use of various animal models, includingnon-human transgenic animals that have been engineered to have specificdefects, or carry markers that can be used to measure the ability of atest compound to reach and affect different cells within the organism.Due to their size, ease of handling, and information on their physiologyand genetic make-up, mice are a preferred embodiment, especially fortransgenic animals. However, other animals are suitable as well,including C. elegans, rats, rabbits, hamsters, guinea pigs, gerbils,woodchucks, cats, dogs, sheep, goats, pigs, cows, horses and monkeys(including chimps, gibbons and baboons). Assays for modulators may beconducted using an animal model derived from any of these species.

Determining the effectiveness of a compound in vivo may involve avariety of different criteria. Also, measuring toxicity and doseresponse can be performed in animals in a more meaningful fashion thanin in vitro or in cyto assays.

VI.) The Role of OSM and Metastasis

The role of inflammation in invasion and metastasis, particularly ofprostate tumors, is not well understood. However, several studies show acorrelation between serum levels of the interleukin-6 (IL-6) familyinflammatory cytokines and distant metastases. Oncostatin M (OSM) is aninflammatory modulator in the interleukin-6 (IL-6) cytokine family thatcan be associated with the metastatic potential of prostate carcinomas.OSM promotes human prostate cancer cell proliferation,epithelial-mesenchymal transition (EMT) and detachment in vitro. Inprostate cancer cells, OSM can induce vascular endothelial growth factor(VEGF) and urokinase-type plasminogen activator (u-PA), two proteinssuspected to be involved in tumor progression. In addition, a pattern ofincreased OSM expression can occur in high grade Gleason (aggressive)carcinomas. OSM's influence on prostate cancer has only been minimallystudied in vitro. We determined that OSM promotes invasive capacity ofprostate tumor cells and their metastasizing potential.

Androgen deprivation therapy is the standard course of treatment forprostate cancer. However, most prostate cancers treated through androgendeprivation therapy eventually recur. Moreover, prostate tumors withhigh Gleason scores can be metastatic and/or refractory to androgendeprivation therapy. Therefore, alternative therapies, such as the OSMinhibitors disclosed herein can be advantageous.

OSM can play a role in prostate cancer. OSM expression can be directlyassociated with metastatic potential in clinical prostate carcinoma,with increasing OSM and OSM receptor expression being found in higherGleason grade tumors. In vitro studies have shown that OSM induces bothvascular endothelial growth factor (VEGF) and urokinase-typeplasminogen-activator (u-PA) expression in DU-145 PCa cells, each ofwhich are implicated in tumor progression. In addition, theproliferation of the DU-145 and 22Rv1 prostate cancer cell linesincreases with increasing OSM values. OSM can also induce the epithelialto the mesenchymal transition (EMT), cell detachment, and invasivecapacity of DU-145 cells.

OSM has been implicated in tumor invasion and metastasis of other tumorsas well. These other types of tumors can include, for example, ovarianand breast cancer. Hereinafter and in Examples 1-8, the inventorsdescribe their detailed findings in relation to breast cancer, as theseresults can be readily extended to the understanding and treatment ofprostate cancer.

We have studied the role of Oncostatin M (OSM) promoting the metastasisof breast tumors. OSM is a pro-inflammatory pleiotropic IL-6 familycytokine that plays a role in development, neurogenesis, liverregeneration, and haematopoiesis. OSM activates signaling pathways bybinding its receptors, OSM receptor (3 (OSMRβ) or leukemia inhibitoryfactor receptor β (LIFRβ) dimerized with a common gp130 subunit. OSMalso activates the JAK/STAT, MAPK, and PI3K/AKT pathways via binding itsreceptors. Moreover, OSM activates the stress-activatedmitogen-activated protein kinases p38 and JNK.

In vitro studies examining the role of OSM signaling in breast cancerhave suggested that this cytokine may increase the metastatic potentialfor breast epithelial cells. While OSM inhibits proliferation of variouscarcinomas including lung cancer, multiple myeloma and breast cancer, inother cancer types it actually increases proliferation. Though OSM'seffect of tumor cell proliferation is not the same for any carcinoma,recent research suggests that this cytokine can promote invasion andmetastasis. Specifically, OSM can function on various cancer cells invitro to: 1) promote the transition from an epithelial to mesenchymalphenotype (EMT), 2) upregulate expression of proteases such ascathepsins, and matrix metalloproteinases (MMPs), 3) promote tumorcell-substrate invasion and detachment, and 4) induce the expression ofvascular endothelial growth factor (VEGF), HIFla and other proangiogenicfactors, 5) induce increased cancer cell stemness, suppress ER alphaexpression, and promote S100A7 expression.

A role for OSM produced by tumor-associated immune cells can promote ametastatic phenotype in breast cancer cells in vitro. In response tobreast cancer cells in vitro, macrophages and neutrophils collected fromhealthy human volunteers can express and secrete high levels of OSM,thereby supporting a role for OSM in the breast tumor microenvironment.Additionally, breast cancer cells alone secrete high levels of OSM inculture in addition to the immune cells. Taken together, these in vitrostudies suggest a role for both autocrine and paracrine signaling by OSMin tumor metastatic potential, particularly during early stages of themetastatic cascade.

Transgenic and other mouse models of OSM can show its importance in theproduction of red blood cells and platelets, wound-healing in the heartand liver, bone homeostasis, inflammatory cell migration andinfiltration into injured tissue, and leukocyte activation.

We discovered that in vivo OSM can increase mammary tumor metastases tobone and increase bone metastatic osteolysis. Thus, OSM can influencenormal bone homeostatis and in the bone metastatic microenvironmentduring later stages of breast cancer metastasis. Increased OSMexpression can also lead to changes in ECM remodeling during breasttissue involution.

More specifically, we investigated the effect of OSM on early stages ofbreast cancer metastasis. First, we established the expression patternof OSM in human breast tissue using tissue microarrays. We showed thatOSM is expressed at higher levels in Invasive Ductal Carcinoma (IDC)than the adjacent normal breast tissue, but at highest levels in ductalcarcinoma in situ (DCIS), suggesting that OSM may play an important rolein the early stages of breast tumor invasion and metastasis. In order tofurther investigate this feature in vivo, we utilized the syngeneicmodel of 4T1.2 mouse mammary tumor cells that when injected into Balb/cmice established a metastatic pattern similar to that seen in breastcancer patients. We showed that a reduced OSM expression in 4T1.2 cells(4T1.2-shOSM) is sufficient to inhibit the progression and final numberand volume of metastases in the lung. In this orthotopic model, ourresults demonstrate that reduced OSM significantly increases survivalpost-primary tumor resection; however, bypassing the early stages ofmetastasis by injecting cells directly into the systemic circulation,did not. We confirmed OSM increases early stage metastatic potential invitro by showing OSM induces 4T1.2 tumor cell detachment and migration.These findings were corroborated with an in vivo study demonstratingthat when OSM is injected peri-tumorally in a mice harboring MDA-MB-231mammary tumors, there is an increase in the number of circulating tumorcells as well as spontaneous metastasis to lung. Findings from thesestudies suggest that autocrine and paracrine OSM in the tumormicroenvironment acts as a potent initiator of invasion and the earlystages of metastasis. Therefore, modification of OSM levels in the tumormicroenvironment can be a highly effective therapeutic strategy forhalting the invasion and metastasis of breast cancer.

An intriguing finding of this study is that OSM epithelial expression ishigher in earlier stages of ductal carcinoma of the breast (DCIS andIDC) compared to metastatic disease, which is consistent with theproposed role of OSM in vitro that it promotes the initiation ofinvasiveness of breast cancer. Higher levels of OSM and OSMR are seen inbreast tissue as it progresses from ductal carcinoma in situ to invasiveductal carcinoma. Although OSM expression has been studied in varioushuman tumors such as prostate cancer, ovarian cancer and keratocanthoma,the present study may provide additional insights into the role of OSMin breast cancer and in these other types of cancers. In breast cancer,in vivo research suggests a role for OSM in metastasis. OSM can promotebone metastases and bone destruction from the metastases and can promotethe protein expression of EMT markers.

We investigated the role of OSM during the earliest stages of invasionand metastasis in an animal model. Our orthotopic injection studies haveshown that OSM promotes maximum metastatic burden in lung and bone.Additionally, utilizing a modified human breast cancer cell line, theMDA-MB-231 cells, in an athymic, immunocompromised mouse, OSM applieddirectly to the tumor microenvironment increases the number ofcirculating tumor cells and number of metastasis to the lung. However,when we bypass the early stages of metastasis or those precedingcolonization at a secondary metastatic site by implanting 4T1.2 mousemammary tumor cells via intracardiac injection, there is no change inmetastasis to the lung or survival time driven by OSM.

The difference between using a Balb/c mouse with mouse mammary tumorcells versus an athymic mouse with human breast cancer cells is notinsignificant and one major difference between the two models.Application of human OSM directly to the tumor microenvironment did notaffect primary tumor growth with the human MDA-MB-231 cells-DH3LN-luc.,while loss of OSM from the mouse cells increased tumor growth. Anexplanation for this could be that full-length OSM may be less effectiveon growth inhibition in vivo due to its need for processing and itsdifferential ability to signal when bound to proteins in theextracellular matrix of the tumor micron environment. Human OSM geneencodes a 252 amino acid (aa) polypeptide: the first 25 aa encode asignal sequence for secretion; the remaining 227 aa are the precursorprotein named pro-OSM or full-length OSM. Cleavage of the c-terminis ata trypsin-like cleavage site yields the mature (aka active or truncated)196 aa form. Both full-length and truncated OSM display similar receptorbinding affinity, but full-length OSM was found to be 5 to 60-fold lesseffective on growth inhibition. Processing of OSM may regulate OSMactivities in vivo. This differential growth effect between the twoforms explains why peri-tumor injection of full-length OSM had no effecton primary tumor growth, but still maintained the functional propertiesof increased metastatic burden.

To explain the role of OSM in early stages of metastasis, we showed that4T1.2 cells can detach, invade, and migrate in response to the cytokine.Epithelial cells, from which the majority of cancer cells such as 4T1.2cells arise from, are not normally motile or invasive. In order forcancer cells to detach, invade, and become mobile through the ECM, theyare thought to transform from an epithelial phenotype to a moremesenchymal phenotype. This phenomenon known as EMT involvesreorganization of the cytoskeleton to allow cells to become mobile andthus migrate. As the cells become mesenchymal, they can produceproteinases such as matrix metalloproteinases to detach and invade intothe ECM. Our results suggest that OSM's activity is primarily focusedwithin the primary tumor and does not have an effect once the cellsescape into the circulatory system. This is seen as OSM promotes tumordetachment and invasion in vitro, and metastasis in vivo when breasttumor cells are injected orthotopically.

It has been thought that tumor cell EMT, detachment, and invasionpromote tumor cell propagation into the circulation and are thought tobe precursors for intravasation and circulating tumor cells (CTC).Specifically, the cell's propensity to intravasate into circulationdirectly correlates to the level of CTC's. CTC's have also been linkedclinically to enhanced metastatic burden in patients and a reduced5-year survival rate. While the exact mechanisms that regulate CTCgeneration is unclear, factors such as transforming growth factor beta(TGF-beta), IL-6, and interleukin-8 may act as promoters of CTCs eitherby increasing tumor cell invasion, detachment, or EMT. Additionally,CTCs in general can tend to have a heterogeneous expression of genes andmixed epithelial/mesenchymal phenotypes, which complicate detection, andprognostic prediction. Results suggest that tumor cells that intravasateinto circulation and become CTCs undergo EMT, invade through the ECM,and detach. Our data shows that OSM injection increases the number ofCTCs in tumor bearing mice and this increase may be caused by OSM'seffects on the early stages of metastases. Inflammatory cytokines suchas IL-6, IL-17, and M-CSF at the site of the secondary lung metastasis,as well as in systemic circulation may foster their ability toproliferate, survive, and migrate. To date, it is not believed thatinvestigation of OSM as a cellular cue in primary breast tumorenvironment promoting metastasis to the lung, despite several studies ofthe very closely related IL-6 cytokine.

OSM's pivotal role in the metastasis of primary cancers of the breast tosites such as bone and lung, and the known properties of OSM in theprogression of inflammatory diseases, including arthritis and lungfibrosis give insight into the mechanisms that might drive OSM in themetastatic cascade. Tumors often appear to have chronic inflammationmediated by tumor-associated macrophages (TAMs). TAMs generally exhibitan M2 phenotype in which they produce growth factors and cytokines,promote ECM remodeling through the expression of proteases, and increaseangiogenesis through the production of VEGF. Both tumor-infiltratingmacrophages and neutrophils express and release OSM in response tobreast carcinoma cells in vitro. OSM can also play a role in the switchfrom the M1 phenotype to M2 phenotype. OSM can also be both pro andanti-inflammatory in vivo but in the absence of tumor cells, OSM cantrigger inflammatory cell recruitment when expressed in joints andlungs, and enhance dendritic cell homing to regional lymph nodes. Inaddition, OSM can promote changes in the ECM by increasing collagendeposition and increasing expression of proteases. In arthritic models,OSM can promote bone and cartilage production at higher levels thanIL-6. Inflammatory cytokines such as IL-6, IL-17, and M-CSF can promotemammary tumor metastasis to the lung in the PyV MT mouse model when anarthritic disease state is induced with Type II collagen. In studies ofskin inflammation, OSM can contribute to keratinocyte hyperplasia inpsoriasis and dermatitis.

OSM can also promote inflammation and fibrosis in lung tissue andcurrently, and if chronic inflammation in the lung is present fromeither smoking or disease, breast cancer metastasis to the lung may bepromoted. In both our in vivo models with either decreased OSMexpression (4T1.2 mouse mammary tumor cells in a healthy Balb/c mouse)or application of OSM directly to the tumor microenvironment (MDA-MB-231human breast cancer cells in an immunocompromised nude mouse), there isno increased basal inflammation in the lung microenvironment. Therefore,we can assume that inflammation is not the driving force for ourobserved differences in metastatic burden by OSM. However, OSM appearsto be more important for initiating the early steps in the metastaticcascade preceding colonization in the lung.

OSM also increased IL-6 expression only in the ER-breast cancer celllines MDA-MB231, MDA-MB-468, and 4T1.2 cells. IL-6 can increase breastcancer migration, invasion, and detachment. IL-6 can also induces EMT,and high serum IL-6 levels can be associated with poor prognosis inbreast cancer patients. OSM's effect on tumor progression may also bemagnified by the induction of IL-6, as IL-6 can also promote furtherinduction of IL-6 production in a feed forward system.

In the bone microenvironment, OSM expression by metastatic cells candirectly interfere with the homeostasis of the osteogenic cells, andlead to increased osteolysis, suggesting a role for OSM in themetastatic niche during later stages of metastasis. Returning toprostate cancer, the inventors now describe their further findings inrelation to this tumor. FIG. 10 shows a schematic of a mechanism bywhich OSM is presumed to promote the metastasis and progression ofprostate tumors. OSM produced by prostate tumor cells promotes theexpression of proteases, such as MMPs and cathepsins that in turnstimulate EMT, detachment, and invasiveness of tumor cells. In addition,OSM can induce tumor cell expression of proangiogenic factors such asVEGF and u-PA that promote angiogenesis and metastasis.

OSM can play a role in the initial stages of prostate cancer metastasis.DU-145 prostate cancer cells can undergo increased proliferation, EMT,detachment, and invasive potential in response to OSM treatment (seeFIGS. 11-14). Initial results suggest that OSM's effects onproliferation and cell detachment work through a STAT3 signalingmechanism, but that its effects on invasive potential may work through adifferent pathway. In vitro studies indicate that OSM can inducevascular endothelial growth factor (VEGF) and urokinase-type plasminogenactivator (u-PA) in prostate cancer cells.

Given the role of OSM in the metastases of prostate cancer and othertypes of cancer, we investigated various small molecule inhibitors ofOSM. It is our assumption that increased inhibition of OSM may becorrelated with a decreased degree of cancer metastases, particularlyprostate cancer metastases. Several classes of small molecules wereinvestigated in this regard and are summarized in Table 1, Formulas 1-10below along with their in silico predicted binding constants for OSM.

TABLE 1 AutoDock Predicted Binding OSM Free Binding Energy ConstantChemical Class Structure Compound ID (kcal/mol) (uM) bis(benzimidazole)(1)

NCI 61610 −9.33 0.14 spirocyclopropane (2)

CB_CL 181230 −8.56 0.53 spirocyclopropane (3)

CB_CL 81250 −8.39 0.71 spirocyclopropane (4)

CB_CL 111696 −8.54 0.55 quinolone (5)

Florida ZINC 15777366 −8.27 0.87 spirocyclopropane (6)

CB_CL 19531 −8.44 0.65 dihydronaphthalene- 2-one (7)

Florida ZINC 15770835 −7.47 3.35 benzochromene (8)

Florida ZINC 32603276 −7.53 3.02 naphthyridine-2-one (9)

CB_CL 12813 −7.30 4.46 phenanthridine-6- one (10)

NCI 127133 −7.12 6.04

Methods for inhibiting OSM can include exposing OSM or a cell lineexpressing OSM to a small molecule inhibitor of OSM, including those ofFormula 1-10 or various derivatives thereof.

Methods for treating cancer can include administering a pharmaceuticalcomposition containing a small molecule OSM inhibitor to a patient withprostate cancer. Administration can be orally, parenterally, rectally,topically, intravenously, and the like under a suitable dosing schedulecompatible with the metabolic clearance of the active compound. Theactive compound is included in a pharmaceutically acceptable carrier inan amount sufficient to exert a therapeutically useful effect in thepatient being treated. The therapeutically effective concentration maybe determined empirically by testing the compounds in in vitro and invivo systems described herein and then extrapolated therefrom fordosages for humans. The concentration of active compound in thepharmaceutical composition will depend on absorption, inactivation andexcretion rates of the active compound, the physicochemicalcharacteristics of the compound, the dosage schedule, and amountadministered as well as other factors known to those of skill in theart.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Patent publications citedherein and the materials for which they are cited are specificallyincorporated by reference. To facilitate a better understanding of theembodiments of the present disclosure, the following examples are given.In no way should the following examples be read to limit, or to define,the scope of the invention.

EXAMPLES

Tissue Microarrays: Breast tissue was obtained from paraffin blockarchives at the Department of Pathology, Mercy Medical Center, Nampa, IDand removed of patient identity as per IRB guidelines. Three tissuemicroarrays (TMA) of 1 mm thickness and totaling 72 patients were madeusing a Quick-Ray, an instrument used for boring tissue from a paraffinblock (Woo-Ri Medic, Kent, Wash.). Two blocks consisted of tissues from54 breast cancer patients (32 adjacent normal, 9 DCIS patients, 54 IDC)without metastasis and included three primary tumor cores and oneadjacent normal core for each case. The third block included samplesfrom a total of 18 breast cancer patients (18 adjacent normal, 3 DCIS,18 IDC and 16 metastatic) with lymph node metastasis and contained threeprimary tumor cores, two metastatic cores and one adjacent normal tissuecore per case. The TMAs included a row of control tissues includingspleen, lung, placenta, salivary gland, liver and brain. Spleen andsalivary gland served as positive controls for OSM staining.

Immunohistochemistry: The TMAs were stained for oncostatin M using theHistostain Kit (Cat #95-9843; Invitrogen, Carlsbad, Calif.) permanufacturer's instructions. The TMAs were deparafinized using Histosol(National Diagnostics, Atlanta, Ga.) and stained overnight with 1:400dilution of rabbit anti-human OSM primary antibody (Cat #sc-129; SantaCruz Biotechnology, Santa Cruz, Calif.) and 1 hour with 1:1000 goat-antirabbit IgG-AP secondary antibody. TMAs stained with secondary antibodyalone served as the negative control, and spleen and salivary glandserved as positive controls for OSM staining. The specificity of thea-OSM antibody was tested by treating tissue sections with 10-times theamount of OSM blocking peptide (Santa Cruz Biotechnologies, Santa Cruz,CA) and incubating overnight at 4° C. The mixture was then diluted tothe required concentration and immunohistochemistry was performed asabove.

The TMAs were analyzed for OSM expression and the intensity of OSMstaining in the ductal epithelium, stroma, and blood vessels of adjacentnormal and cancerous tissue was graded as follows: 0=No staining;1=Light staining; 2=Medium staining; 3=Dark staining. In order toconfirm reproducibility of the results, the pathologist reread 10 TMAcores that were chosen randomly, and his observations were consistentwith the previous results. In cases where a single core had bothcancerous and normal tissue, the OSM expression data in the cancerouspart was combined with other cancerous tissues and the expression in thenormal part was combined with other adjacent normal tissues forstatistical analysis. Also in these cases, stroma, and blood vesselswere considered as cancerous.

The TMAs were also stained with CD15 antibody that specifically stainshuman neutrophils and CD68 antibody that specifically stains humanmacrophages. Tissues were deparafinized, hydrated and treated with 3%hydrogen peroxide solution for 10 min, treated with a target antigenretrieval solution in a pressure cooker for 15 min, rinsed, andincubated with the primary antibody (CD15 or CD68) for 30 min. This wasfollowed by secondary antibody staining for 15 min, DAB staining for 10min and hematoxylin staining for 10 min. The above staining procedurewas conducted at the Mercy Medical Center, Nampa, ID using anautostainer (DAKO, DC3400-7218-03). By comparing the macrophage orneutrophil staining to the corresponding OSM staining, we visuallyassessed if the macrophages or neutrophils expressed OSM.

Statistical analysis: Assessments from multiple cores for each patientwere averaged for each cell tissue type (ductal epithelial, vessel,stroma) and stage of malignancy (normal, DCIS, IDC, metastatic) that waspresent in the core. The four stages were statistically compared amongductal tissues. For vessels and stroma, the stage was characterizedeither as cancerous or non-cancerous because the three cancerous stages(DCIS, IDC, metastatic) could not be ascertained for these tissues. TheOSM staining intensity was analyzed as a mixed model to accommodaterepeated observations on each patient. These repeated observations wereassumed to have non-negligible correlation and were modeled understandard repeated measures variance-covariance assumptions. Stage wastreated as a fixed effect. Additionally, statistical models consideringpatient prognostic markers were evaluated. These models included thestage and the prognostic marker, with or without an interaction; themodel with the lowest AIC_(c) was selected to determine whether theprognostic factor was associated with OSM staining. These prognosticfactors considered were age, tumor size, lymph node status,angiolymphatic invasion, tumor grade, tumor type, histologic grade,nuclear atypia, margin status, mitotic rate, Her2/neu expression,progesterone and estrogen receptor. Initial assessments indicated thatOSM staining intensity did not differ significantly between the twogroups of patients (patients with and without lymph node metastasis) sopatient group was not included as an analysis factor in the study. Allmodels were assessed for adequacy by residual analysis, a concern herebecause of the bounds on OSM staining intensity (0-3) and our specificinterest in changes in mean staining intensities. No predicted valuesexceeded the possible observational boundaries and residual patternswere acceptable despite the categorical nature of the data collection.All analyses were conducted using SAS version 9.1.3 (SAS 2004).

Cell lines and culture conditions: 4T1.2 cells were cultured in MEMamedia supplemented with 10% fetal bovine serum (FBS), 1 mM sodiumpyruvate, and 100 units/ml each of penicillin and streptomycin, andpassaged for no more than 6 months. MDA-MB-231 D3H2LN luc2 cells(Caliper Life Sciences) were cultured in RMPI media supplemented with10% FBS and 100 units/ml of penicillin and streptomycin. Cells weremaintained at 37° C., 5% carbon dioxide, and 95% humidity. All media andsupplements were obtained from Hyclone (Logan, Utah). 4T1.2 mousemammary cell lines were generated in-house.

Plasmid construct design and cell transfections: To create OSM knockdownvectors, OSM shRNA and a LacZ shRNA sequences were cloned into thepSilencer 4.1 plasmid (Ambion, Austin, Tex.). Briefly, 4T1.2 cells weretransfected with the pSilencer 4.1 constructs containing the one of thetwo mOSM shRNAs or a LacZ control shRNA using lipofectamine LTX(Invitrogen Carlsbad, Calif.) reagent, as per manufacturer instructions.Stably transfected cell lines were grown in the presence of 0.3mg/mL ofthe neomycin analogue G418 (Sigma Aldrich). All established cell lineswere checked for OSM expression by ELISA.

Animals and Tumor Cell Injections: For syngeneic studies, six-week-oldfemale Balb/c mice were obtained from the National Cancer Institute'sAnimal Production Facility (Frederick, MD). For orthotopic injections,each mouse was anesthetized by i.p. injection of 6.25 mg/kg of sodiumpentobarbital or with 2.5% isoflurane and 1.0×10⁵ cells diluted in 10 μLof PBS containing 10% medium were injected into the 4^(th) mammary fatpad. For tumor resection, mammary tumors were surgically excised 14 daysafter orthotopic injections. All animal studies were conducted inaccordance with the protocol approved by the Institutional Animal Careand Use Committee (IACUC) at the Boise VA Medical Center, WashingtonUniversity School of Medicine in St. Louis, Mo., or the Peter MacCallumCancer Centre in Melbourne, Australia. Starting at 2 weekspost-injection, tumor length and width were measured by mechanicalcalipers 3 times a week and tumor volume was extrapolated using thefollowing equation (tumor volume=(length×widtĥ2)/2). “Survival endpoint”was defined by the IACUC as tumor size greater than 20 mm in diameter,10% or more weight loss, and/or appearance of cachexia.

For xenograft models, six-week-old female nude mice were obtained fromthe National Cancer Institute's Animal Production Facility (Frederick,Md.). Non-surgical orthotopic injections were performed using 2.0×10⁶cells diluted in 50 μL of PBS containing 10% medium. When the tumorsbecame palpable, mice were randomized into groups and began receivingper-tumoral injections. For peri-tumoral injections, either 50 μL PBS or1 μg recombinant full length human OSM (Peprotech) diluted in 50 μL PBSwas injected into the area surrounding the tumor three times per weekuntil the end point of the experiment.

Quantitative PCR (qPCR): For quantitative analysis of lung metastases,lungs dissected from mice bearing mammary tumor were snap-frozen inliquid nitrogen and pulverized into a fine powder. DNA was extractedusing an STE buffer containing 20 μl/ml of proteinase K and purified bytwo phenol/chloroform (1:1 v/v) extractions followed by ethanolprecipitation. The ratio of cancer cells to normal cells was quantifiedby measuring the neomycin resistance gene (neon) DNA levels versus thevimentin DNA loading control. Taqman PCR was performed on an AppliedBiosystems 7500 real-time thermocycler. The cycling conditions were runas follows: 50° C. for 5 minutes, 95° C. for 2 minutes, then 40 cyclesof 95° C. for 1 minute and 60° C. for 45 seconds. Fluorescence wasmeasured every cycle after the annealing step and threshold cycle number(CO values were calculated. The data was analyzed using the comparativeDCt method.

Detection of CTCs: Detection of human circulating tumor cells (CTCs) inmouse blood was performed. Human DNA standard curve was prepared byadding a specified number of human MDA-MB-231 cells into mouse blood andthe DNA isolated for use in the qPCR reactions. Whole DNA was isolatedfrom 100 μl of whole blood collected from mice at the end of theexperiment. DNA was isolated using DNeasy Blood & Tissue kit (QuiagenCat#) using the manufacturer's standard instructions. DNA concentrationswere normalized between each sample and 4.5 ng of DNA was added to each25 μl qPCR reaction. qPCR reaction mixture was obtained from the GoTaqqPCR Master Mix (Promega, Cat#TM318, Madison Wis.), and reactionmixtures were prepared in accordance with manufacturer recommendations.0.125 μl of 100 μM primers for human Alu fragment, and for GAPDH wasadded to each reaction. The primer sequences used for Alu was(s:CACCTGTAATCCCAGCACTTT a:CCCAGGCTRGGAGTCGCAGT) and for

GAPDH the sequence used was (s: ATGACATCAAGAAGGTGGTG; a:CATACCAGGAAATGAGCTTG). The qPCR reaction was done with SYBR greenchemistry using a CXR reference dye and ran on an Applied BiosystemsAB7300 real-time thermocycler. Reaction conditions were: 50° C. for 2minutes, 95° C. for 3 minutes and 40 cycles of: (95° C. 00:15, 60° C.00:30, 72° C. 00:30) and fluorescence measurements were taken during theannealing temperature stage (60° C.). cT values were determined and thefinal results were normalized to GAPDH signal levels.

In-Vivo Magnetic Resonance Imaging: Respiratory-gated, spin-echo MRimages of mice were collected in an Oxford Instruments (Oxford, UK) 4.7tesla, 40-cm bore magnet. The magnet was equipped with Agilent/Magnex(Yarnton, UK) actively shielded, high-performance (21-cm inner diameter,˜30 G/cm, ˜200 ms rise time) gradient coils and International ElectricCompany (Helsinki, Finland) gradient power amplifiers and interfacedwith an Agilent/ Varian NMR Systems (Santa Clara, CA) DirectDrive™console. All data were collected using a Stark Contrast (Erlangen,Germany) 2.5 cm birdcage if coil. Prior to the imaging experiments, micewere anesthetized with isoflurane and were maintained on isoflurane/O₂(1-1.5% v/v) throughout data collection. Animal core body temperaturewas maintained at 37±1° C. by circulation of warm air through the boreof the magnet. During the imaging experiments, the respiration rates forall mice were regular and ˜2 sec⁻¹. Synchronization of MR datacollection with animal respiration was achieved with arespiratory-gating unit and all images were collected duringpost-expiratory periods. Imaging parameters are TR=3 s, TE=20 ms,FOV=2.5 cm², Data matrix=128×128; slice thickness=0.5 mm; number ofaverages (NEX)=4.

In vivo bioluminescence imaging (BLI) and tumor progression: BLI of liveanimals was initiated at 13 days after cell line injection and performedweekly. Three to five mice were imaged at one time. Ex vivo organs werealso imaged using BLI. Both procedures follow our previously describedprotocols.

Histology: To verify lung metastasis, lungs from each experimental group(4T1.2-LacZ, n=2; 4T1.2-shOSM2, n=2; and 4T1.2-shOSM1, n=1) were placedin ultralight fixative (Ultralight Histology, Nampa, Id.) paraffinembedded, and sectioned (Bi-Biomics, Nampa, Id.). For each spine, four 1μm sections were collected 10 μm apart in the lumbar region and H&Estained.

IL-6 ELISA: 1×10⁵ T47D, MDA-MB-231, MCF7, MDA-MB-468, or 4T1.2 cellswere plated in multi-well plates in the presence or absence of OSM for47 hours. Conditioned media was collected, diluted 1:5 and an IL-6 ELISAwas performed as according to the manufacturer's recommendedinstructions.

3 mm cubes were excised from tumors dissected from mice injected withMDA-MB-231 cells. The tissue was homogenized in lysis buffer (Pathscanlysis buffer, Cell Signaling) using a pestle. Cell lysates were thendiluted 1:5 and IL-6 levels in the lysates were analyzed by ELISA asdiscussed above.

Statistical Analysis. Data are displayed as mean ±standard error of themean (SEM). Data were analyzed using an unpaired student's t-test oranalysis of variance (ANOVA) with Tukey's multiple comparison post-hoctest where appropriate, using Prism GraphPad 5.0b software (GraphPadSoftware Inc., San Diego, Calif.). Survival data were analyzed using theLog-rank (Mantel-Cox) test. In the analyzed data, asterisks denote*p<0.05, **p<0.01, or ***p<0.001.

Example 1

Oncostatin M is highly expressed in early stages of ductal carcinoma ofthe breast. The expression of OSM in a series of TMA breast samples wasanalyzed by immunohistochemistry (IHC). Treatment of OSM antibody with10-fold excess OSM blocking peptide followed by IHC resulted in nopositive staining, indicating that the antibody is specific to OSM (datanot shown).

Three TMAs containing samples from a total of 72 patients were employedfor this study. Of these, 54 patients were from the non-metastatic groupand 18 were from the metastatic group. The metastatic group containedsamples from patients diagnosed with metastasis of IDC to lymph nodes.All patients in this study were diagnosed with IDC. A total of 12patients also had DCIS. In addition, the TMAs contained adjacent normaltissues from a total of 50 patients (Table 1).

Table 1 shows how the 72 patients provided data for the statisticalanalysis of OSM expression. All of the 72 samples with IDC expressed OSMwhile 46 of the 50 adjacent normal ductal tissues were positive for OSMexpression. OSM staining intensity differed significantly among the fourstages (F_(3,278)=10.0, p<0.001). Adjacent normal breast tissue meanstaining intensity (1.33) was significantly less than that of DCIS(2.00) and IDC (1.66) (FIG. 1A; Table 1). In these tissues, OSMexpression was uniformly concentrated in the cytoplasm of the ductalepithelial cells with some expression in the stroma and blood vessels(discussed in the following section). Additionally, OSM expression inthe metastatic tissue (1.24) was lower than that of IDC and DCIS, whileit was statistically similar to adjacent normal breast tissue. Ductalepithelial cells of cancerous breast tissue express high levels of theOSMRβ and gp130 subunits of the OSM receptor; therefore, OSM expressionby the ductal epithelial cells indicates an autocrine mechanism for OSMfunction in breast cancer progression.

Our finding that OSM expression is higher in earlier stages of breastcancer than in metastatic tissue suggests that OSM may promote theinvasive capacity of breast tumors in vivo and its role may be lesssignificant once the tumor has metastasized to distant tissues. It isbelieved that OSM-promoted cell detachment and invasive capacity isthrough the induction of several tumor promoting factors such as COX-2,MMPs, cathepsins-D and -L, basic-fibroblast growth factor (bFGF), andVEGF. This pattern of OSM expression is also consistent with an EMT(epithelial to mesenchymal transition), which has previously beensuggested as a role for OSM. Since metastatic tissue contains tumorcells that have already been altered and need to resume proliferation,OSM may no longer be required and hence may be down regulated. Recentfindings have shown that OSM is not highly expressed in pure, isolatedcases of in situ ductal carcinoma, which usually does not have invasivecapacity. OSM is also highly expressed in mature keratocanthoma that hasa tendency to involute and infiltrate breast tumors.

Example 2

OSM promotes mammary tumor metastasis to lung. To examine whether OSM isrequired for mammary tumor metastasis to lung, we undertook stableknockdown of OSM expression. Two independent OSM shRNA sequences werecloned into the pSilencer4.1 vector and transfected into 4T1.2 mousemammary tumor cells (T1.2-shOSM1 and 4T1.2-shOSM2) using a procedureknown to result in a 3 to 12-fold reduction in OSM expression. To testthe effects of OSM on mammary tumor metastasis in vivo, control4T1.2-LacZ, 4T1.2-shOSM1, and 4T1.2-shOSM2 cells were injectedorthotopically into the mammary fat pads of Balb/c mice. Low levels ofsecreted OSM in the 4T1.2-shOSM2 cells were shown by our lab to resultin increased primary tumor growth. However, injection of 4T1.2-shOSM1cells, which displayed a modest decrease in tumor cell-secreted OSM, didnot affect tumor growth in vivo.

Metastasis to lung in mice injected with control 4T1.2-LacZ,4T1.2-shOSM1, and 4T1.2-shOSM2 cells were quantified by qPCR. Mean lungmetastatic burden in lung was 10-fold lower in mice that received4T1.2-shOSM1 cells and 5-fold lower in mice injected with 4T1.2-shOSM2cells, compared to 4T1.2-LacZ control cells (FIG. 1A). Histologicalevaluation of lung by H & E staining revealed the presence of largemetastases in mice injected with 4T1.2-LacZ control cells but many fewerand smaller metastases in mice injected with 4T1.2-shOSM1 and4T1.2-shOSM2 cells (FIG. 1A). Additional histology performed on tissuesfrom mice injected with parental 4T1.2 cells, using an anti-mouse OSMantibody, showed strong OSM expression in the primary mammary tumor aswell as some background expression in the normal breast connectivetissue (FIG. 1B). Very high OSM expression was shown at the leading edgeof the primary tumor metastasis, in closest proximity to the breaststroma (FIG. 1B). In total, these results suggest that OSM is necessaryfor spontaneous mammary tumor metastasis to lung and may be aided byinteractions with the lung tissue inflammatory microenvironment.

Example 3

OSM expression increases the number and volume of lung metastases invivo. To more specifically characterize and quantify the progression oflung metastases seen in vivo after injection of parental 4T1.2, control4T1.2-LacZ, and 4T1.2-shOSM2 cells, in vivo magnetic resonance imaging(MRI) experiments were performed. Respiratory-gated spin-echo images,with coronal orientation, were collected with sufficient slices (e.g.,21 slices, 0.5 mm thickness) to completely cover the lungs of eachanimal. Mice were imaged at early (days 20-21), mid (days 25-26), andlate (days 29-30) stages of in vivo metastasis (FIG. 3A). For all threecell types, MRI spectra showed essentially no detectable metastasis atthe early stages. At mid and late stages, however, readily identifiablemetastases were observed in lung images. Lung tumors were manuallysegmented with IMAGE J (rsbweb.nih.gov/ij), and the number and volume ofall metastatic tumors were measured and recorded, on an animal-by-animalbasis. As illustrated in FIG. 2B, the number of metastases was reducedby more than 50% in mice injected with 4T1.2-shOSM2 cells compared tocontrol 4T1.2-LacZ control cells at both mid and late stages ofmetastasis. Additionally, the average metastasis volume wassignificantly decreased by 50 to 80% in 4T1.2-shOSM2 cells compared to4T1.2-LacZ control or parental 4T1.2 cells, respectively, at mid andlate stages of metastasis. Thus, the in vivo MRI imaging suggested thatOSM is a potent inducer of the metastatic cascade that results in lungmetastases originating from a primary mammary tumor.

Example 4

Lack of mammary tumor cell-produced OSM increases survival fromspontaneous metastasis via orthotopic injection but not via intracardiacinjection in vivo. To determine the influence of decreased OSMexpression on metastasis independent from its effects on primary tumorcell proliferation, we utilized a tumor resection model. Orthotopicmammary fat pad injections were performed using control 4T1.2-LacZcells, 4T1.2-shOSM1, and 4T1.2-shOSM2 cells, primary tumors wereresected at day 14 (FIG. 4A), and mice were monitored until endpointcriteria were met (see above). The mean survival time of the mice thatreceived 4T1.2-shOSM1 and 4T1.2-shOSM2 cell injections significantlyincreased by a mean of 37 and 42 days and a maximum of 44 and 72 days,respectively, compared to 4T1.2-LacZ that survived a mean of 32 days andmaximum of 36 days. These results suggest that following primary mammarytumor resection, decreased OSM expression in primary tumor cells leadsto a delay in metastasis and increased survival.

In order to determine if OSM expression is most important in the earlyor late stage of metastasis, we injected the mammary tumor cellsdirectly into the systemic circulatory system via the left ventricle ofthe heart. Intracardiac injections of control 4T1.2-LacZ and4T1.2-shOSM2 cells did not result in significant changes in survivaltime or the number of cells that colonized in the lungs (FIG. 4B). Thisindicates that OSM expression in tumor cells affects the early stages ofmetastasis that proceed their successful survival in circulating bloodmore significantly than later stages of metastasis involvingcolonization, survival, and proliferation at distant metastatic sitessuch as lung.

Example 5: OSM affects in vivo using human MDA-MB-231 D3H2LN cells. Todetermine the influence of OSM in human breast cancer, we used axenograft model.

MDA-MB-231 D3H2LN luc2 cells were injected into the fourth mammary fatpads of female nude mice. After the tumors were palpable, OSM (1 μg) orPBS were injected peri-tumorally 3 times per week (FIG. 6A), and micewere monitored until the end-point criteria were met. Tumor size wasmeasured using calipers three times per week for the duration of theexperiment and expressed as tumor volume (mm³)=(length×width). Tumorvolume did not differ between the groups (FIG. 6B). Next, BLI intensityof the cells was assessed in vivo. The BLI intensities of the tumorsfrom both groups were similar with variations in intensities in bothgroups (FIG. 6C). A few mice in both groups had lower BLI intensitiesdue to tumor necrosis.

Example 6

OSM increases the metastatic volume within the lungs which correlateswith an increase in circulating tumor cells. Ex vivo analysis of thelungs was performed. Mice receiving peri-tumoral OSM injections showedlarger metastatic volume while the mice receiving PBS injections showeda few micro-metastases within the lungs (FIG. 6C). Interestingly, lungsof two of the OSM injected mice were imaged on an order of 10⁶photons/sec/cm² (FIG. 6C; OSM group, right panel) while the other lungswere imaged on an order of 10⁴ photons/sec/cm² (FIG. 6C; PBS group andOSM group, left panel). Next, quantification of the BLI intensities ofthe lungs was performed. Results showed that lungs extracted from theOSM injected group were two orders of magnitude (10²) higher than thePBS injected group.

Our studies on detecting CTCs involve the usage of the Alu transposonrepeats for detecting human cells in mouse blood, and with colonyforming assays that detects all transformed cells in the blood. Weverified this by measuring the number of circulating tumor cells (CTCs)in mice orthotopically injected with mammary tumor cells. In order todetect circulating tumor cells in the mouse circulatory system, DNA wasisolated from mouse blood, and human alu fragment concentrations weredetermined in the blood by qPCR. In our xenograft model, we injectedhuman MDA-MB-231 cells into the 4^(th) mammary fatpad and anycirculating tumor cells contain multiple copies of human Alu fragments.In animals that received rhOSM injections, there was a 3.5 fold increasein the number of circulating tumor cells per 100 μl of mouse blood overanimals that did not receive any OSM injections.

Example 7

OSM promotes IL-6 expression in ER negative cell lines. OSM induced IL-6expression was studied on various cell lines including two ER+celllines, T47D and MCF7, and three ER- cell lines MDA-MB-468, MDA-MB-231,and 4T1.2 cells. rhOSM was used for the four human cell lines at aconcentration of 25 ηg/ml while rmOSM was used for the 4T1.2 cells at aconcentration of 25 ηg/ml. The cells were incubated in OSM for a totalof 48 hours and IL-6 levels in the conditioned media was assessed byELISA. OSM did not induce IL-6 on the ER+MCF7 or T47D cells, while inMDA-MB-468 cells, OSM induced IL-6 approximately 5-fold, while inMDA-MB-231 cells IL-6 levels increased by approximately 4-fold. In the4T1.2 mouse mammary cancer cells, OSM induced IL-6 by approximately4-fold (FIG. 7). To test if OSM induced IL-6 in vivo, tumors from theMDA-MB-231 injected mice were lysed, homogenized, and the IL-6 levelswere analyzed by ELISA.

Example 8

OSM promotes metastases to the spleen and liver in a mouse model. Asshown in FIG. 9, 4T1.2 metastases were observed in the spleen and liverin a mouse model.

Example 9

Proliferation and detachment of prostate cancer cells. Prostate cancercell lines DU-145 and PC-3 were plated in triplicate in complete RPMImedia at a cell density of 1,000 cells/ml and allowed to adhereovernight. Cells were treated the next day with OSM at a concentrationof 17.5 ηg/ml (untreated cells were used as a control). Both detachedcells and total cells were counted on days 1, 3, 5, 7, and 9. Detachedcells were collected, stained with trypan blue and viable cells werecounted using a hemocytometer. Adherent cells were trypsinized, stainedwith trypan blue and viable cells were counted on a hemocytometer. Tolook at the effects of OSM on prostate cancer cell proliferation, thenumber of viable detached cells and adherent cells were added togetherto determine the total number of cells.

The addition of OSM significantly increased proliferation (FIG. 11)(P=0.0247) and cell detachment (FIG. 12) (P=<0.0001) in DU-145 cells,but not in STAT3 PC-3 cells. These results suggest that OSM mediates itseffect on proliferation and detachment through a STAT3 signalingpathway.

The Epithelial-Mesenchymal Transition (EMT) was also measured. Duringthe proliferation and detachment assays detailed above, micrographs ofthe DU-145 cells were taken at day 5 before cells were collected andcounted. At day 5, untreated DU-145 cells exhibit an epithelialmorphology and were packed closely together in a round colony formation(FIG. 13). However, the OSM-treated DU-145 cells showed a moremesenchymal morphology, were more fibroblastic in shape, and spreadfurther apart from each other.

Invasion potential was also measured. OSM increased the invasionpotential of both DU-145 and PC-3 cell lines (FIG. 14). Twenty threepercent of treated DU-145 cells showed an invasive potential as comparedto 15% of untreated DU-145 cells, and 21% of treated PC-3 cells showedan invasive potential as compared to 14% of untreated PC-3 cells. PC-3cells pretreated with OSM for 72 hours had a 4-fold increase in invasivepotential, as compared to untreated PC-3 see FIG. 15. These resultssuggest that the increased invasive potential mediated by OSM uses asignaling pathway distinct from that of proliferation and celldetachment and one that does not use STAT3.

Example 10

Our preliminary calculations show that there are three potential SMIbinding sites on the OSM surface. A structural alignment of OSM andLIF-LIFR complex (PDB ID: 2Q7N) indicates that site 1 is putativelyclose to OSM-OSMR binding interface, site 2 is located at the waistregion of OSM, and site 3 is at the far end from the binding interface.SMIs that bind to site 1 are expected to directly interrupt OSM bindingto OSM receptor. SMIs that bind to site 2 or site 3 may also interferewith OSM-OSM receptor binding through allosteric effects. Structuresidentified by AutoLigand will be used to query compounds from anin-house database using the OpenEye suite program (41). AutoDock

Vina and AutoDock 4.2 programs will be used to rank drug-like compoundsbased on their predicted binding free energy potential to binding sites.

TABLE 2 Compounds with favorable OSM interaction at Site 1 obtained byAutoDock 4.2 Program. AutoDock Predicted Binding Binding CompoundCompound Free Energy Constant Number ID (kcal/mol) (uM) OSM-SMI-1NSC21357

−6.88 9.11 OSM-SMI-2 NSC81514

−6.04 27.52 OSM-SMI-3 NSC105360

−8.45 0.64 OSM-SMI-4 NSC112821

−5.81 55.05 OSM-SMI-5 NSC348965

−7.93 1.53 OSM-SMI-6 NSC382916

−8.99 0.26 OSM-SMI-7 NSC636120

−8.26 0.88 OSM-SMI-8 NSC642624

−7.56 2.87 OSM-SMI-9 NSC645072

−7.03 7.05 OSM-SMI-10 NSC647257

−9.86 0.06 OSM-SMI-11 NSC648596

−5.87 50.04 OSM-SMI-12 NSC127133

−7.12 6.04 OSM-SMI-13 NSC61610

−9.33 0.14 OSM-SMI-14 CB_CL111696

−8.54 0.55 OSM-SMI-15 CB_CL19531

−8.44 0.65 OSMI-SMI-16 CB_CL81250

−8.39 0.71 NSC: National Cancer Institute Diversity Set III, CB_CL:ChemBridge Combinational Libraries.

Table 2 along with CB_CL111696, CB_CL19531, and CB_CL81250 from Table 1show the top candidates obtained from our initial preliminary virtualscreening for interactions at Site 1 of three databases containing˜345,000 compounds. We plan to screen a total of 2.5 million compoundsfrom multiple select databases. All compounds with predicted bindingconstants in the <10 μM range and/or binding free energies higher than−5.0 kcal/mol will advance.

Off-target effects will be assessed by computational prediction of leadOSM-SMI for their specificity to OSM, as compared to other IL-6cytokines including LIF, interleukin-11 (IL-11), ciliary neurotropicfactor (CNTF), and cardiotrophin-1 (CT-1). Compounds with favorablepredicted binding constants and no predicted off-target effects willalso be assessed using the SciFinder program to identify related or“like” compounds. Chemicals showing greater than 70% relatedness and notidentified in our in silico screening of 2.5 million compounds will feedback into the initial in silico screening. Overall, the top 100compounds that exhibit substantial selectivity and specificity will beconsidered viable drug leads and undergo in vitro testing.

Next, we will evaluate the in vitro performance of the top OSM-SMI leadsobtained from in silico screening. This involves testing OSM-SMIs forsignal inhibition and in vitro efficacy, respectively.

All human breast carcinoma cell lines used in this aim will be purchasedfrom the American Type Culture Collection (ATCC) or Caliper LifeSciences, and the growth of frozen cell aliquots will be initiated on aperiodic basis to remove the possibility of cross-cell contamination.The cell lines will be regularly tested for mycoplasma contamination byboth DAPI staining and PCR. We will use four invasive ductal carcinomacell lines [T47D, MCF7, MDA-MB-231 and MDA-MB-468]. These include theless aggressive, estrogen receptor-positive (ER+), progesterone receptorpositive (PR+), HER2-, luminal T47D and MCF7-luc cell lines, as well asthe triple negative breast cancer (TNBC) basal-like cell line,MDA-MB-231-LN-luc, that is metastatic in orthotopic xenografts and theless metastatic TNBC cell line MDA-MB-468. All cell lines express OSMRb,LIFRb, and gp130, as well as various levels of OSM, by RT-PCR (data notshown).

Next an enzyme-linked immunosorbent assay (ELISA) for analyzing initialOSM-induced phosphorylation of STAT3 on Tyr-705 (pSTAT3) will beemployed for the initial screen. T47D, MCF7, and MDA-MB-231 human breastcancer cells induced pSTAT3 upon stimulation with OSM (25 r_(i)g/m1) for30 minutes. The ability of the top 100 OSM-SMIs to inhibit OSM-OSMRinteractions that result in expression of pSTAT3 will be evaluated bythis assay and cells will be pretreated with the OSM-SMIs for 2 hoursprior to the addition of OSM. As positive controls, neutralizingantibodies to human OSM or human gp130 will establish baseline pSTAT3levels in the ELISA assay (FIG. 16A).

Using this pSTAT ELISA test, we preliminarily screened 16 identifiedlead compounds (Table 1) in T47D and MDA-MB-231 breast cancer cells(FIG. 8B, C) and Du145 human prostate cancer cells (FIG. 8D) anddemonstrated a significant inhibition of pSTAT3 with OSM-SMI-8 aftertreatment with a reduced level of OSM (5 ηg/ml). Actual physiologicallevels of OSM may be much lower than the nanogram levels used here.Healthy humans have been shown to have OSM serum levels of 6-13 ρg/ml,cancer patients with hepatocellular carcinoma have serum levels of39-121 pg/ml, and mice with MDA-MB-231 mammary tumors contain 6 to 85ρg/ml in their serum (data not shown). Therefore, testing inhibitorsusing lower concentrations of OSM may better allow for the detection ofeffects at physiologically relevant concentrations.

Additionally, a functional dose response curve for OSM-SMI activity willbe generated through 2 log units of concentration of compounds and usedto establish the dynamic range of OSM-SMI activity. This will be used todetermine the half maximal inhibitory concentration (IC₅₀) as well asthe minimum effective concentration (Ceff) for each compound. Inpreliminary data, OSM-SMI-8 demonstrated an IC₅₀ of 531 ηM when testedin MDA-MB-231 cells after treatment with OSM (5 ηg/ml) for 30 minutes(FIG. 9A).

The top 20 positive OSM-SMI compounds (IC₅₀<5 μM) will be assessed forthorough blocking of OSM signaling. OSM-SMIs will be introduced to T47D,MCF7, MDA-MB-231, and MDA-MB-468 cells at the determined C_(eff) andtested for inhibition of pSTAT3, pJNK, pERK, and pAKT by Western blotanalysis. In preliminary data, OSM-SMI-8, -10 and -11 (5 μM) were testedfor suppression of downstream signaling by Western blot analysis of twoindependent sets of MDA-MB-231 cell lysates (FIG. 17B). This experimentconfirmed the ELISA test for OSM-SMI-8 suppression of pSTAT3, as well asdemonstrating inhibition of pJNK, pERK, and pAKT. OSM-SMI-10 and -11also inhibited signaling to various extents (FIG. 9B). This datasuggests that the in silico screening is successful in identifyingOSM-SMIs that can block OSM signaling.

OSM-SMIs will also be biologically evaluated for off-target effects bytheir specificity for OSM and not other IL-6 cytokines. The four humanbreast cancer cell lines [T47D, MCF7, MDA-MB-231, and MDA-MB-468] willbe pretreated with each OSM-SMI for 2 hours and then treated with IL-6,LIF, IL-11, CNTF, or CT-1 (5 μM) for 30 minutes. Cell lysates will beevaluated by Western blot analysis for pSTAT3, pJNK, pERK, and pAKT.

The top 10 candidates will be evaluated for their ability to inhibittumor cell proliferation, cell detachment, vascular endothelial growthfactor (VEGF) secretion, and invasive capacity of the above mentionedcell lines by addition of OSM-SMIs to cell culture media at C_(eff).These experiments will proceed in a fashion similar to our previouslypublished work analyzing human and mouse mammary carcinoma cells invitro. Briefly, proliferation will be measured by cell counting afterpretreatment of cells with each OSM-SMI and then treatment with OSM for2, 4, and 6 days, and percent-detached cells will be determined overtime by counting the number of non-adherent cells and dividing thisnumber by the total number of cells. To examine OSM-induced invasivepotential in vitro, Matrigel invasion assays (BD Biosciences) will beperformed as described previously. VEGF secretion will be assessed inthe conditioned media of cells pretreated with the OSM-SMIs and thentreated with OSM for 48 hours.

The steady state drug dissociation constants, KD, of the candidateOSM-SMIs will be determined. Binding studies will be performed in thepresence of OSM using LC-MS technique incorporating a C18 reverse phaseHPLC column to effect separation and detection of the unbound OSM-SMIfraction.

To assess cellular toxicity of the top 5 OSM-SMIs against human breastcancer cells, dose-response curves for cell viability will beestablished for the above mentioned cell lines, as well as normal humanmammary epithelial cells (HMECs), using CellTiter-Glo (Promega) andLC₅₀/LC₉₀ values established. To evaluate global toxicity andorgan-specific toxic effects, the integrated discrete multiple organcell culture (IdMOC) system developed by Li will be used. Compounds witha 100-fold or greater normal/tumor cell toxicity ratio and an IdMOC cellsurvival rate above 90% will be advanced for in vivo testing.

Next, we will perform in vivo testing of candidate OSM-SMIs with theexplicit purpose of identifying one or more compounds that can inhibitmetastasis in a mouse model of human breast cancer.

The top 3 to 5 compounds will be pre-screened for acute toxicity.Briefly, 50 mg/kg of each compound will be dissolved in vehicle[dimethyl acetamide (DMA)/PEG-400/PBS] and injected intravenously intothe tail vein of three mice. Mice will be observed for signs of acutetoxicity over 48 hours. Up to three OSM-SMIs exhibiting no toxicity willenter the pre-clinical studies.

In vivo studies will be performed using the MDA-MB-231-LN-luc orthotopicxenograft mouse model of breast cancer. Female athymic mice will receive10⁶ tumor cells in 10 μl PBS injected into the 4^(th) mammary fat pad atday zero, and randomized into different groups. Tumors will be resectedwhen they reach 5 mm in diameter. After tumor resection, animals willreceive candidate drugs at three different concentrations in vehicle byintraperitoneal (i.p.) injections three times per week for the life ofthe animal. Conservative estimates from our preliminary data on OSMknockdown indicate that a group size of n =20 mice will have 80% powerat α=0.05 to detect differences in survival of 10 days and >80% power todetect difference in tumor growth and metastasis of at least 20%.

Whole blood will be analyzed for complete blood counts (CBC), and serumwill be analyzed for OSM and IL-6 levels. CTCs, tumor growth, andmetastasis will be measured.

End point metastases will be evaluated by ex vivo BLI for all metastaticsites as well as micro-CT for bone. Histological analysis will beperformed to confirm the mammary carcinomas and metastases, andimmunohistochemistry (IHC) of relevant cytokines (OSM and IL-6) will beperformed to characterize the invasive tumor edge.

Statistical Analysis: Statistical analysis will be performed with theassistance of our biostatistician, Laura Bond, MS, at Boise StateUniversity. In vitro experiments will be performed at least intriplicate with analysis by t-test and ANOVA with a priori multiplecomparisons. Effects on organ specific and total metastatic burden, CTCnumbers, tumor growth, and time to metastasis will be assessed byMANOVA. Conservative estimates from our preliminary data indicate thatthe proposed group size will give 80% power to detect differences intotal metastatic burden of at least 20% at α=0.05. Greater power isanticipated for CTC numbers and tumor growth. The Kaplan-Meier test willbe used to compare survival times between groups.

Example 11

1. Protein Oncostatin M Genbank

AAH11589 (SEQ ID NO: 1) MGVLLTQRTL LSLVLALLFP SMASMAAIGS CSKEYRVLLGQLQKQTDLMQ DTSRLLDPYI RIQGLDVPKL REHCRERPGAFPSEETLRGL GRRGFLQTLN ATLGCVLHRL ADLEQRLPKAQDLERSGLNI EDLEKLQMAR PNILGLRNNI YCMAQLLDNSDTAEPTKAGR GASQPPTPTP ASDAFQRKLE GCRFLHGYHRFMHSVGRVFS KWGESPNRSR RHSPHQALRK GVRRTRPSRK GKRLMTRGQL PR BC011589.1(SEQ ID NO: 2) 1 gtcaccccca gcgggcgcgg gccggagcac gggcacccagcatgggggta ctgctcacac 61 agaggacgct gctcagtctg gtccttgcac tcctgtttccaagcatggcg agcatggcgg 121 ctataggcag ctgctcgaaa gagtaccgcg tgctccttggccagctccag aagcagacag 181 atctcatgca ggacaccagc agactcctgg acccctatatacgtatccaa ggcctggatg 241 ttcctaaact gagagagcac tgcagggagc gccccggggccttccccagt gaggagaccc 301 tgagggggct gggcaggcgg ggcttcctgc agaccctcaatgccacactg ggctgcgtcc 361 tgcacagact ggccgactta gagcagcgcc tccccaaggcccaggatttg gagaggtctg 421 ggctgaacat cgaggacttg gagaagctgc agatggcgaggccgaacatc ctcgggctca 481 ggaacaacat ctactgcatg gcccagctgc tggacaactcagacacggct gagcccacga 541 aggctggccg gggggcctct cagccgccca cccccacccctgcctcggat gcttttcagc 601 gcaagctgga gggctgcagg ttcctgcatg gctaccatcgcttcatgcac tcagtggggc 661 gggtcttcag caagtggggg gagagcccga accggagccggagacacagc ccccaccagg 721 ccctgaggaa gggggtgcgc aggaccagac cctccaggaaaggcaagaga ctcatgacca 781 ggggacagct gccccggtag cctcgagagc accccttgccggtgaaggat gcggcaggtg 841 ctctgtggat gagaggaacc atcgcaggat gacagctcccgggtccccaa acctgttccc 901 ctctgctact agccactgag aagtgcactt taagaggtgggagctgggca gacccctcta 961 cctcctccag gctgggagac agagtcaggc tgttgcgctcccacctcagc cccaagttcc 1021 ccaggcccag tggggtggcc gggcgggcca cgcgggaccgactttccatt gattcagggg 1081 tctgatgaca caggctgact catggccggg ctgactgcccccctgccttg ctccccgagg 1141 cctgccggtc cttccctctc atgacttgca gggccgttgcccccagactt cctcctttcc 1201 gtgtttctga aggggaggtc acagcctgag ctggcctcctatgcctcatc atgtcccaaa 1261 ccagacacct ggatgtctgg gtgacctcac tttaggcagctgtaacagcg gcagggtgtc 1321 ccaggagccc tgatccgggg gtccagggaa tggagctcaggtcccaggcc agccccgaag 1381 tcgccacgtg gcctggggca ggtcacttta cctctgtggacctgttttct ctttgtgaag 1441 ctagggagtt agaggctgta caaggccccc actgcctgtcggttgcttgg attccctgac 1501 gtaaggtgga tattaaaaat ctgtaaatca ggacaggtggtgcaaatggc gctgggaggt 1561 gtacacggag gtctctgtaa aagcagaccc acctcccagcgccgggaagc ccgtcttggg 1621 tcctcgctgc tggctgctcc ccctggtggt ggatcctggaattttctcac gcaggagcca 1681 ttgctctcct agagggggtc tcagaaactg cgaggccagttccttggagg gacatgacta 1741 atttatcgat ttttatcaat ttttatcagt tttatatttataagccttat ttatgatgta 1801 tatttaatgt taatattgtg caaacttata tttaaaacttgcctggtttc taaaaaaaaa 1861 aaaaaaaaa

Therefore, the present disclosure is well adapted to attain the ends andadvantages mentioned as well as those that are inherent therein. Theparticular embodiments disclosed above are illustrative only, as thepresent invention may be modified and practiced in different butequivalent manners apparent to those skilled in the art having thebenefit of the teachings herein. Furthermore, no limitations areintended to the details of construction or design herein shown, otherthan as described in the claims below. It is therefore evident that theparticular illustrative embodiments disclosed above may be altered,combined, or modified and all such variations are considered within thescope and spirit of the present invention. The invention illustrativelydisclosed herein suitably may be practiced in the absence of any elementthat is not specifically disclosed herein and/or any optional elementdisclosed herein. While compositions and methods are described in termsof “comprising,” “containing,” or “including” various components orsteps, the compositions and methods can also “consist essentially of” or“consist of” the various components and steps. All numbers and rangesdisclosed above may vary by some amount. Whenever a numerical range witha lower limit and an upper limit is disclosed, any number and anyincluded range falling within the range is specifically disclosed. Inparticular, every range of values (of the form, “from about a to aboutb,” or, equivalently, “from approximately a to b,” or, equivalently,“from approximately a-b”) disclosed herein is to be understood to setforth every number and range encompassed within the broader range ofvalues. Also, the terms in the claims have their plain, ordinary meaningunless otherwise explicitly and clearly defined by the patentee.Moreover, the indefinite articles “a” or “an,” as used in the claims,are defined herein to mean one or more than one of the element that itintroduces. If there is any conflict in the usages of a word or term inthis specification and one or more patent or other documents that may beincorporated herein by reference, the definitions that are consistentwith this specification should be adopted.

1-11. (canceled)
 12. A method of inhibiting migration of metastatictumor cells or neutrophils to a pre-metastatic organ comprisingadministering to the subject an effective amount of a OSM or OSMreceptor (gp-130) antagonist.
 13. The method of claim 12, wherein thepre-metastatic organ is lung or liver.
 14. The method of claim 12,wherein the OSM antagonist is a small molecule.
 15. The mL thod of claim8 wherein said small molecule is one from table 1 or
 2. 16. The methodof claim 9 wherein said small molecule is OSM-SMI-8,:
 17. The method ofclaim 12 wherein the OSM antagonist is an anti-OSM antibody or afragment thereof.
 18. The method of claim 12 further comprisingadministering to the subject an effective amount of a chemotherapeuticagent.
 19. The method of claim 12, wherein the subject is human.
 20. Akit of parts for use in inhibiting or reducing tumor cell detachment,proliferation and/or metastasis, comprising an OSM antagonist, and anadministration vehicle.
 21. A pharmaceutical composition comprising aunit dose of at least 1 mg, of an antagonist to oncostatin M (OSM), anda pharmaceutically acceptable carrier.
 22. The pharmaceuticalcomposition according to claim 21 wherein the antagonist is combinedwith a chemotherapy agent.
 23. The pharmaceutical composition of claim21 wherein an OSM antagonist is a small molecule selected from compoundslisted in Table 1 or
 2. 24. The pharmaceutical composition of claim 23wherein said small molecule is one or more of OSM-SMI-8, OSM-SMI-10, orOSM-SMI-11.
 25. The pharmaceutical composition of claim 21, wherein saidcomposition is effective for inhibiting or reducing the spread of aprimary tumor to a pre-metastatic organ of the subject.
 26. Thepharmaceutical composition of claim 21, wherein the antagonist iscombined with an effective amount of gp-130 antagonist.
 27. Apharmaceutical composition for use in inhibiting or reducing tumor celldetachment, proliferation and/or metastasis, comprising an effectiveamount of an antagonist to OSM, wherein the OSM antagonist is one ormore of OSM-SMI-8, OSM-SMI-10, or OSM-SMI-11 and a pharmaceuticallyacceptable carrier.
 28. The pharmaceutical composition of claim 26,wherein the OSM antagonist is combined with a chemotherapy agent, animmune modulator or an anti-OSM antibody or a fragment thereof.
 29. Thepharmaceutical composition of claim 27, wherein reducing tumor celldetachment, proliferation and/or metastasis comprises reducing size ornumber of lung metastases.